Background The effective dose during ultrasound-guided rectus sheath block (URSB) for reducing pain after laparoscopic tubal ligation is reported to be 100 mg of 0.25% bupivacaine. We examined the minimal effective dose of 0.25% bupivacaine for URSB on oral analgesic requirement after ambulatory single-port laparoscopic tubal resection. Methods A prospective, randomized controlled, superiority trial was conducted among patients who had been scheduled for ambulatory laparoscopic tubal resection between September 2015 and January 2019 at a tertiary care hospital in southern Thailand. Anesthesia was induced following protocol. The intervention group was allocated to receive a bilateral URSB using 10 ml of 0.25% bupivacaine on either side after intubation (total 50 mg) while the control group did not receive the sham block. Patients and assessors were blinded to the study intervention. All patients received a multimodal analgesia regimen as follows: fentanyl and ketorolac intraoperatively and fentanyl and oral acetaminophen at the post-anesthetic care unit. Postoperative oral analgesic requirement (acetaminophen and/or ibuprofen) at home was the primary outcome. Postoperative time to first analgesic requirement, oral analgesia (acetaminophen/ibuprofen), and pain score at 6 and 24 h were accessed via telephone interviews. Percentage, effect size (ES), and 95% confidence interval (CI) were presented. Results A total of 66 out of 79 eligible patients were analyzed (32 intervention, 34 control). Intraoperative fentanyl consumption was significantly lower in the intervention group (ES [95% CI]: 0.58 [0.08, 1.07] mcg, p = 0.022). Time to first oral analgesia in the intervention group was significantly longer than that of the control group (ES [95% CI]: 0.66 [0.14, 1.16] h, p = 0.012). The proportion of oral analgesia requirement at 24 h after surgery in the control group was significantly higher than that in the intervention group (97% vs 75%, p = 0.012). Pain scores at 6 and 24 h were similar in both groups although slightly lower in the intervention group (ES [95% CI]: 0.22 [−0.26, 0.71], p = 0.368 and 0.33 [−0.16, 0.81], p = 0.184, respectively). Conclusion A dose of 0.25% bupivacaine 50 mg for URSB reduced the oral analgesic requirement at 24 h and prolonged the time to first analgesic requirement after ambulatory laparoscopic tubal resection. Trial registration Thaiclinicaltrials.orgTCTR20150921002. Registered on 18 September 2015