The mucosal barrier that separates the microbiota from the nervous system is disrupted during gastrointestinal (GI) inflammation. It is presently unknown whether translocation of bacteria during GI inflammation modulates visceral sensation and thereby contributes to pain. The primary aim of this study was to determine if a defined community of 33 commensal GI microbes (MET-1) alters the excitability of mouse dorsal root ganglion (DRG) neurons. To determine whether supernatant containing the secretory products of MET-1 altered the excitability of DRG neurons, and identify the intracellular signaling pathways involved. In addition, we sought to identify candidate bacterial mediators of this effect. DRG neurons were dissociated from C57Bl6 male mice and incubated overnight in sterile MET-1 supernatant (1:10 – 1:10,000 dilution in sterile media) or sterile control media, in the presence or absence of various inhibitors. Current and voltage clamp experiments were performed after 24 hours. MET-1 decreased the excitability of DRG neurons in a concentration-dependent manner by significantly increasing rheobase. 1:100 MET-1 was used in subsequent experiments. The resting membrane potential of DRG neurons was hyperpolarized by MET-1, -68 mV (n=7; MET-1) vs. -58 mV (n=30; controls), p < 0.05; Mann-Whitney test. In addition, MET-1 increased voltage-gated K+ current (p<0.001; 2 way ANOVA). Addition of a nuclear factor kappa B (NFκB) inhibitor (SC-514, 20 mM) or an ERK1/2 inhibitor (PD 98059, 30 mM) blocked the effects of MET-1. A bacterial protease inhibitor cocktail (1:10,000) abrogated MET-1 effects on DRG neurons. The serine protease inhibitor (FUT-175, 50 mM), but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases, abolished the effects of MET-1. The serine protease, cathepsin G (100 nM) recapitulated the effects of MET-1 on the excitability of DRG neurons. Blocking protease activated receptor (PAR) 2 (GB83, 10mM) or PAR4 (P4pal10, 10mM) did not block the effects of MET-1 on the excitability of DRG neurons. Serine proteases secreted by MET-1 can directly impact the function of DRG neurons, through NFκB and ERK1/2-dependent pathways. On the basis of these observations, pain signaling may be modulated by microbiota-neuronal interactions.