Abstract Background Abdominal pain is a primary symptom of inflammatory bowel disease (IBD). Opioids provide relief from IBD-associated pain, but they are addictive and associated with excess mortality in IBD patients. Thus, there is a need to develop novel therapeutics for IBD-associated pain. The mechanosensitive ion channel, transient receptor potential melastatin 3 (TRPM3) is upregulated in sensory neurons innervating inflamed tissue and contributes to pain from inflamed joints and cystitis. However, TRPM3’s role in abdominal pain has not been investigated. Purpose To evaluate whether TRPM3 contributes to abdominal pain using a mouse model of colitis. Method We used ratiometric Ca2+ imaging and extracellular afferent nerve recording to determine the effects of pharmacological activation or inhibition of TRPM3 on T13-L5 dorsal root ganglia (DRG) neurons and lumbar splanchnic nerves, respectively. Increased intracellular Ca2+ indicates neuronal excitation. Furthermore, the effects of TRPM3 activation in neurons and nerves from healthy mice and mice with dextran sulphate sodium-induced colitis were compared. Result(s) The TRPM3 agonists, CIM-0216 (0.1-10µM) and pregnenolone sulphate sodium (PSS; 1-300µM), concentration-dependently increased intracellular Ca2+ concentration in mouse DRG neurons and this was blocked using the TRPM3 inhibitor isosakuranetin (5µM; p<0.0001, Mann-Whitney Test). CIM-0216 (5µM)-induced increases in intracellular Ca2+ were significantly larger in neurons from mice with colitis (326±16% of baseline) compared to neurons from healthy mice (257±13% of baseline; p<0.01, Kruskal-Wallis with Dunn’s Multiple Comparison Test). The percentage of neurons responding to CIM-0216 was significantly increased in mice with colitis compared to healthy mice (79% vs 62%; p<0.001, Fischer’s Exact Test). Similarly, the percentage of neurons responding to PSS from mice with colitis was increased compared to healthy mice; however, this did not reach statistical significance (75% vs 70%, p=0.351, Fischer’s Exact Test). Furthermore, CIM-0216 (20µM)-induced change in the basal firing of lumbar splanchnic nerves was significantly increased in mice with colitis (1.23±0.24Hz) compared to healthy mice (0.60±0.14Hz, p<0.05, unpaired t-test). Conclusion(s) TRPM3 activation excited DRG neurons and lumbar splanchnic nerves and these excitatory effects were augmented in mice with colitis. Disclosure of Interest None Declared