Circulating levels of TMAO are elevated in humans with cardiometabolic diseases. The gut microbiota-initiated TMA/flavin-containing monooxygenase 3 (FMO3)/TMAO pathway has been identified as a potential modulator of host cardiometabolic phenotypes. Like TMA/TMAO, FMO3 expression is positively correlated with body mass index and high-fat diet-induced obesity We hypothesized that TMA/TMAO stimulate vascular contraction which could be modified by perivascular adipose tissue (PVAT) expression of FMO3. Our model was the isolated thoracic aorta with (+) or without (-) PVAT, in presence (+) or in absence (+) of endothelium (E) of male Sprague Dawley rat to perform tissue bath studies to measure isometric tone using a large range of TMA/TMAO concentrations: 1 nM-10 -1 M. Immunohistochemistry (IHC) studies were done to identify the presence of FMO3 in aorta. TMA and TMAO, at any concentration, did not relax half maximally contracted tissues when compared to vehicle control. TMA and TMAO elicited arterial contraction independent of PVAT or E status. Interestingly, contractions stimulated by TMA were significantly higher (% PE max 123.7±15.6%) than by TMAO (% PE max 38.3±15.5%). IHC studies revealed FMO3 in aortic PVAT, but the FMO3 inhibitor methimazole (100 μM) did not affect aortic contraction stimulated by TMA (+PVAT). The L type Ca2+ channel antagonist nifedipine (100 nM), reduced the TMA-induced contraction (% PE max: +PVAT: 73±9%; -PVAT: 63±6.7%), compared to vehicle (+PVAT: 149.8±18%; -PVAT: 111.8±9%). These results support that TMA can induce arterial contraction with higher potency and efficacy compared to TMAO, independent of the conversion to TMAO. The contractile mechanism is ≈ 50% dependent of Ca 2+ influx through L-type VOC. Considering the high concentration needed to achieve the contraction it is unlikely that increasing vascular tone is a mechanism that could contribute to CVD but dependence of contraction on Ca 2+ channel activation suggests specific activity of TMA in the vessel wall that warrants further study.