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Using pesticides in the An Giang province is widespread. However, studies on the health effect of organophosphate pesticide have not been updated within the past 12 years. This study aimed to assess exposure to organophosphate pesticides and their effects on sperm quality among farmers in the An Giang Province, Mekong Delta, Vietnam. During the winter – spring crop season of December 2021 to February 2022, a cross-sectional study was conducted on farmers aged 18 to 60 years-old based on seven communes' health checkup programs. The pesticide spray group included farmers who had sprayed pesticides in the previous week and had a history of working in agriculture for more than 2 years. The control group was defined as those who lived in the same community, had not worked in agriculture, and had never sprayed pesticides. Demographic characteristics and blood, urine, and semen samples were collected and analyzed. Data for 184 eligible participants were analyzed, including 116 farmers in the pesticide spray group and 68 non-farmers in the control group. Pesticide spray contributed to a decrease in the sperm quality index of 6.253 units (95% CI, 4.472–8.749). Increasing each pseudocholinesterase (PChE) unit (kIU/L) was associated with an increase of 1.181 units in the sperm quality index (95% CI, 0.757–0.947). Preventive methods for organophosphate pesticide exposure, such as administrative controls, engineering controls, substitution, and personal protective equipment should be applied to control health risks. In the An Giang setting, personal protective equipment is feasible, but most types of equipment are not used. The immediate priority is to determine the cause of personal protective equipment not being used and to find solutions to encourage people to use them. [ABSTRACT FROM AUTHOR]