--- - |2+ Abstract Aim The microbiota shows diurnal oscillations that are synchronized by the host's circadian clock and feeding rhythms. Short‐chain fatty acids (SCFAs) produced by the microbiota are possible synchronizers of peripheral circadian clocks. We aimed to investigate whether faecal SCFAs show a diurnal rhythm that regulates the rhythm of SCFA receptor expression (FFAR2/3, OLFR78, HCAR2) and SCFA‐induced colonic contractility. The role of the circadian clock was studied in mice lacking the core clock gene Bmal1. Methods Mice were sacrificed at 4‐hour intervals. Faecal SCFA concentrations and SCFA receptor expression were determined. The effect of increasing concentrations of a SCFA mix on electrical field‐induced neural responses in colon strips was measured isometrically. Results Diurnal fluctuations in faecal SCFA concentrations (peak 4 hours after lights on) were observed that were in phase with the rhythm of Ffar2/3 expression in the colonic muscle layer. Olfr78 expression was not diurnal and Hcar2 was not detectable. The inhibitory effect of a SCFA mix on neural contractions in colonic smooth muscle strips showed a diurnal rhythm and oscillated in phase with faecal SCFA concentrations and Ffar2/3 expression. In contrast, neither excitatory neural responses nor acetylcholine‐induced smooth muscle contractions showed a diurnal rhythm. In Bmal1−/− mice, no fluctuations in faecal SCFA levels, Ffar3 expression and neural responses to SCFAs were observed. Conclusion Diurnal microbial SCFA levels regulate the rhythm of Ffar3 expression in the colonic myenteric plexus, which causes rhythmicity in SCFA‐induced colonic motility. Deletion of Bmal1 abolishes rhythmicity of SCFA levels and their downstream effects. - 'Acta Physiologica, EarlyView. '