The coordinated transit of intestinal contents is crucial for digestion and host defense, and is regulated by cross-talk between neural circuits, the muscular gut wall and luminal factors. Here we show that androgen signaling to Nos1+ enteric neurons and Scn10a+ spinal afferent neurons is required for normal intestinal transit in mice and is microbiome dependent. Microbial depletion with antibiotics abolished androgen receptor expression in enteric neurons, diminished serum testosterone and caused dysmotility. Androgens were necessary for antibiotics to affect transit and partly sufficient to rescue dysmotility. Nos1 neurons upregulate androgen receptor upon puberty in parallel with shifts in fecal bacterial beta-glucuronidase (GUS) enzymes that can deconjugate steroid glucuronides in mice and humans. Intracolonic administration of a GUS enzyme found to metabolize androgen glucuronides was sufficient to restore neuronal androgen signaling in microbe-depleted mice. Thus, gut microbial reactivation of host-excreted androgens via GUS enzymes represents a dynamic microbe-host interaction that is essential for peripheral nervous system function in homeostasis.