Aurantii fructus immaturus (AFI) and Magnoliae Officinalis Cortex (MOC) have been used to treat constipation in China for thousands of years. In this study, a mouse model of slow transit constipation (STC) was established by gavage of loperamide at a dose of 10 mg/kg bw/day for seven days. Seventy-two mice were randomly allocated to six groups (control, STC model, 3 g/kg AFI + MOC, 6 g/kg AFI + MOC, 12 g/kg AFI + MOC, and mosapride). A mixed aqueous extract of AFI and MOC was administered to the STC mice at the corresponding doses from the first day of modelling. Body weight, faecal water content, gastrointestinal transit time, and intestinal propulsion rate were evaluated. Serum levels of neurotransmitters and gastrointestinal hormones, colonic expression of aquaporins (AQP), and interstitial cells of Cajal (ICC) were assessed using ELISA, immunohistochemistry, and Western blot analysis. The abundance and diversity of the gut microbiota were analysed by 16S rRNA gene sequencing. The mixed aqueous extract significantly increased faecal water content and intestinal propulsion rate and shortened gastrointestinal transit time in STC mice. Furthermore, the administration of AFI and MOC significantly decreased serum vasoactive intestinal peptide (VIP), nitric oxide (NO), and somatostatin (SS) levels and increased serum motilin (MTL) levels in STC mice. The protein expression levels of AQP3 and AQP4 in the colon tissue of STC mice significantly decreased following AFI + MOC treatment, whereas those of AQP9 significantly increased. Moreover, the AFI + MOC treatment led to an increase in the number and functionality of ICCs. In addition, the relative abundances of Ruminococcus and Oscillospira increased in response to the administration of AFI + MOC in STC mice. In conclusion, the mixed aqueous extract of AFI and MOC promoted defaecation and increased intestinal mobility in STC mice. Its mechanisms of action involve modulatory effects on neurotransmitters, gastrointestinal hormones, AQPs, and ICCs. AFI + MOC treatment also improved the diversity and abundance of the gut microbiota in STC mice, particularly short-chain fatty acid-producing bacteria, which may play an important role in its beneficial effect on constipation.