Constipation is one of the most common functional gastrointestinal disorders accompanied with intestinal dysbiosis. Laxatives for constipation usually have side effects. Bee honey is a natural food with unique composition, antimicrobial properties, and bifidogenic effect. In order to assess whether honey can ameliorate loperamide‐induced constipation in BALB/c mice through the alteration of the gut microbiota, the present study was undertaken. Mice were given Jarrah honey (7.5 g/kg body weight) by gavage once per day for 5 days. Fecal water content, intestinal transit rate together with the colon concentrations of substance P (SP), vasoactive intestinal peptide (VIP), and serotonin (5‐hydroxytryptamine; 5‐HT) were evaluated. Furthermore, we determined the effect of honey treatment on gut microbiota in mice using stool genomic 16S rRNA sequencing. As a result, honey showed an obvious improvement in fecal water content and alleviated constipation by modulating the microbial composition of the microbiota, and this was highly associated with a proportional decrease in gut Desulfovibrio. In addition, we found that the colon level of neurotransmitters SP and VIP was significantly related to microbial variations. Our results indicate that gut microbiota is involved in the alleviation of loperamide‐induced constipation by honey supplementation in mice, and it could be considered as an evaluating parameter in constipation therapy strategies.