ScopePeople suffer from constipation caused by many factors, including constipation (Opioid‐Induced Constipation, OIC) during analgesic treatment. Microorganisms may be a potent solution to this problem, but the mechanism is still unclear.Methods and resultsBased on models in vivo and in vitro, the potential mechanism involving Bifidobacterium animalis F1‐7 (B. animalis F1‐7), screened in the previous studies, is explored through non‐targeted metabonomics, electrophysiological experiment and molecular level docking. The results showed that B. animalis F1‐7 effectively alleviates OIC and promotes the expression of chromogranin A (CGA) and 5‐hydroxytryptamine (5‐HT). The metabolite 13,14‐dihydro‐15‐keto‐PGE2 related to B. animalis F1‐7 is found, which has a potential improvement effect on OIC at 20 mg kg BW−1 in vivo. At 30 ng mL−1 it effectively stimulates secretion of CGA/5‐HT (408.95 ± 1.18 ng mL−1) by PC‐12 cells and changes the membrane potential potassium ion current without affecting the sodium ion current in vitro. It upregulates the target of free fatty acid receptor‐4 protein(FFAR4/β‐actin, 0.81 ± 0.02).ConclusionThe results demonstrate that metabolite 13,14‐dihydro‐15‐keto‐PGE2 participated in B. animalis F1‐7 to alleviate OIC via the 5‐HT pathway.