This study was performed to investigate the mechanisms involved in the vasorelaxation induced by mesoionic 2-(4-chlorophenyl)-3-methyl-4-(4-methoxyphenyl)-1;3-thiazolium-5-thyolate (CMMTT), a newly synthesized mesoionic compound, in rat superior mesenteric arteries. In phenylephrine (10 μM)–pre-contracted mesenteric rings, CMMTT (10⁻¹⁴ – 10⁻⁶ M) induced a concentration-dependent relaxation [pD₂ = 10.26 ± 0.05, E_max = 80.8 ± 5.8%], and this effect was almost abolished after either removal of the vascular endothelium [E_max = 17.7 ± 4.2%, P<0.001], removal of the vascular endothelium plus100 μM N^ω-nitro-L-arginine methyl esther (L-NAME) [E_max = 21.0 ± 2.0 %, P<0.001], or after pre-treatment of the rings with 100 μM L-NAME [E_max = 13.3 ± 2.4%, P<0.001] or 10 μM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) [E_max = 13.6 ± 4.8%, P<0.001]. However, endothelium-dependent relaxation induced by CMMTT was not significantly modified after 1 μM indomethacin plus 1 nM atropine [pD₂ = 11.12 ± 0.08, E_max = 73.8 ± 5.15%] or 100 nM charybdotoxin (ChTX) plus 100 nM apamin [pD₂ = 10.89 ± 0.08, E_max = 58.91 ± 9.8%]. In mesenteric rings, CMMTT (10⁻⁶ M) was able to increase nitric oxide (NO)_x levels, and this effect was abolished after removal of the vascular endothelium. In conclusion, the present study, using combined functional and biochemical approaches, demonstrated that CMMTT induced a significant vasorelaxant effect, almost completely mediated by the endothelium, likely via NO release and activation of the NO–cGMP pathway.