Introduction and Objective: Bee venom is a liquid, colorless and acidic mixture (pH 4.5-5.5) that contains 18 different compounds such as enzymes, peptides and biological amino acids. Some of these compounds have anti-inflammatory properties and some have toxic and allergenic properties. Bee venom is produced by female worker bees. The most important peptides in bee venom are melittin, apamin, adolapin and mast cell degranulating (MCD) peptide. The most important enzymes in bee venom can be called hyaluronidase and phospholipase A2 (PLA2). Bee venom has been used in the past, especially in traditional medicine, as a medicine to treat various diseases such as rheumatoid arthritis and also to reduce muscle pain. Massive bee attacks are considered one of the Public Health problems in some countries in the world, including Iran. No specific therapy is available for bee stings and the treatment is done by chemical drugs, leading us to develop Fabbased antivenom as a potential new treatment. So far in Iran, no action has been taken to produce Fab-based antivenom to save people who are sensitive to bee stings. Therefore, the aim of this research was to produce an effective and safe horse serum antidote against the Iranian honey bee (Apis mellifera meda). Material and Methods: Crude bee venom (BV) was prepared from Iranian bees (Apis mellifera meda) using an electric bee venom collecting machine. The poison dries quickly in the air. The dried venom was collected from the glass plates every day with the help of special spatulas and stored in dark glasses in a freezer at -20 ºC for future experiments. To remove mucus and extra substances, the poison is dissolved in a ratio of 1:1 with saline (physiological serum) and centrifuged at high speed. After centrifugation, the upper clear solution is separated and filtered with a 0.2 micron filter. The amount of protein in the crude venom solution was determined by the Bradford protein assay method and based on the standard curve obtained by measuring different concentrations of bovine serum albumin (BSA) solution. The median lethal dose (LD50) value of BV was determined with 24 male mice. Mice were divided into 4 groups. Then 0.5 ml of different doses of venom (60, 70, 80, 90, 100 and 110 μg/ml) dissolved in sterile normal saline were injected intravenously. Control group were injected with 0.5 ml saline solution. Mortality was recorded after 24 hours and LD50 was calculated based on probit analysis. In this research, 3 horses were used to produce antivenom against honey bee venom. For this purpose, horses were immunized with Iranian bee venom 7 times with 7-day intervals, and immunogenicity was evaluated in laboratory conditions with ELISA test. The precipitated antivenom with saturated ammonium sulfate was also used to perform the neutralizing test in mice. To check the quality of antivenom, the neutralization of phospholipase A2 activity was performed using different concentrations of antivenom. Finally, for determining the efficiency of the prepared antivenom, the median effective dose (ED50) value was calculated by probit analysis. In this research, three adult mares (three years old, 400 to 450 kg) were used. To immunize the horses, bee venom was injected with complete and incomplete Freund's adjuvant. Complete Freund's adjuvant was used in the first and second inoculations, and incomplete Freund's adjuvant was used in subsequent inoculations. Venom inoculation was carried out incrementally from 50, 100, 250, 500, 1000, 2000 and 4000 micrograms in 7 times with 7 days intervals. The injection was done subcutaneously (in the neck). Isolation of horse antibody was performed with the standard protocol of the saturated ammonium sulfate method. Evaluation of immunogenicity in laboratory conditions was done by ELISA test. The serum of immunized horses and the precipitated antivenom with saturated ammonium sulfate were was also used to perform the neutralizing test in mice. To check the quality of antivenom, the neutralization of phospholipase A2 activity was performed using different concentrations of antivenom. Finally, for determining the efficiency of the prepared antivenom, the median effective dose (ED50) value was calculated by probit analysis. To determine the effectiveness of the prepared horse antivenom, bee venom was mixed with a constant amount of antivenom in increasing doses and injected into mice. Results: The total protein in the crude BV solution was calculated to be 56 mg/ml. Also, the LD50 of the crude BV was obtained as 4.84 μg/g. ELISA test results showed an immune response that increased more during the immunization schedule. Neutralization of phospholipase A2 activity using different concentrations of antivenom showed that Iranian bee venom has phospholipase activity and 120 μg/ml of this antidote is able to completely neutralize the activity of phospholipase A2. It is clear that, firstly, Iranian honey bee venom has phospholipase activity, and secondly, 55 micrograms of antivenom are needed to neutralize 50% of phospholipase A2 activity in 100 micrograms of venom. The ED50 value will be approximately 54 times the LD50. This means that the antivenom can neutralize up to 54 times the LD50. Therefore, the death caused by 4 mg of bee venom is neutralized by 1 ml of antivenom, so the average effective dose ED50 will be 4 mg/ml. If 100 micrograms of venom enters the body in each bee sting, then 1 ml of produced antivenom can neutralize 40 bee stings. Conclusion: The result showed that horse antivenom against honey bee venom is capable to neutralize the crude venom in mice model. [ABSTRACT FROM AUTHOR]