Acalculous biliary pain may be due to gallbladder dyskinesia or sphincter of Oddi (SO) hypertension. These two etiologies are difficult to differentiate because the gallbladder ejection fraction may be low and the SO manometry results may be abnormal in both. Cholecystectomy is advised for patients with biliary dyskinesia, but it often exacerbates biliary pain for patients with SO hypertension. The biliary pain response to relaxation of the SO using botulinum toxin may indicate appropriate treatment for patients with acalculous biliary pain. The protocol-based management of 25 patients with acalculous biliary pain and two gallbladder ejection fraction estimations less than 40% who had 100 units of botulinum toxin injected into their SO musculature to relax the sphincter has been audited. Patients whose pain was temporarily relieved after botulinum toxin injection were offered endoscopic biliary sphincterotomy, and patients who failed to experience benefit after botulinum toxin injection were assessed for laparoscopic cholecystectomy. Botulinum toxin was injected into the SO of 25 patients, with 11 experiencing temporary biliary pain relief. Of these patients, 10 consented to undergo endoscopic biliary sphincterotomy, with relief of biliary pain in all cases. A total of 14 patients had a negative response to botulinum toxin treatment, with 10 of these patients progressing to laparoscopic cholecystectomy, which resulted in biliary pain relief in eight cases. Botulinum toxin-induced relaxation of the SO may help to direct appropriate therapy for patients with acalculous biliary pain. The data from this study supports the establishment of a randomized clinical trial.