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Abstract Background This study aimed to compare the duration of postoperative analgesia using intraperitoneal bupivacaine plus neostigmine or bupivacaine alone. In this randomized controlled trial, we included 56 adult patients scheduled for elective laparoscopic cholecystectomy operation. Patients were randomly allocated into two groups bupivacaine group (B group) who received 50 ml bupivacaine 0.25% alone and bupivacaine-neostigmine group (BN group) who received 500 μg neostigmine added to 50 ml bupivacaine 0.25%. The study drug was instilled intraperitoneal according to group allocation before the start of the surgery. Primary outcome was the duration of analgesia. Other outcome included total dose of postoperative analgesic consumption and postoperative nausea and vomiting. Results Patients in BN group showed longer duration of analgesia after surgery and longer time for 1st analgesic dose than the patients in B group. Total dose of postoperative pethidine and the incidence of postoperative nausea and vomiting were lower in number in the BN group than in the B group. Conclusions Intraperitoneal instillation of neostigmine as an adjunct to bupivacaine in elective laparoscopic cholecystectomy increased the duration of postoperative analgesia. Also, it might reduce postoperative pain and analgesic requirements as compared to bupivacaine 0.25% alone. Trial registration Name of the registry: Clinical trial Clinical Trial registration number: NCT04244097 Date of registration: 28 January 2020 URL of trial registry record: https://clinicaltrials.gov/ct2/show/NCT04244097