Trimethylamine N-oxide (TMAO), a novel cardiovascular (CV) disease and mortality risk marker, is a gut microbiota-derived metabolite as well. Recently, plasma concentrations of branched-chain amino acids (BCAA) have been reported to be affected by microbiota. The association of plasma TMAO with CV mortality in Type 2 Diabetes (T2D) and its determinants are still incompletely described. We evaluated the association between plasma BCAA and TMAO, and the association of TMAO with CV mortality in T2D individuals. We used data of 595 participants (mean age 69.5 years) from the Zwolle Outpatient Diabetes project Integrating Available Care (ZODIAC) cohort were analyzed. Plasma TMAO and BCAA were measured with nuclear magnetic resonance spectroscopy. CV mortality risk was estimated using multivariable-adjusted Cox regression models. Cross-sectionally, TMAO was independently associated with BCAA standardized (Std) β = 0.18 (95% Confidence Interval (CI) 0.09; 0.27), p <0.001. During a median follow-up of 10 years, 113 CV deaths were recorded. In Cox regression analyses, adjusted for multiple clinical and laboratory variables including BCAA, TMAO was independently associated with CV mortality: adjusted hazard ratio (adjHR) 1.93 (95% CI 1.11; 3.34), p = 0.02 (for the highest vs. the lowest tertile of the TMAO distribution). The same was true for analyses with TMAO as continuous variable: adjHR 1.32 (95% CI 1.07; 1.63), p = 0.01 (per 1 SD increase). In contrast, BCAAs were not associated with increased CV mortality. In conclusion, higher plasma TMAO but not BCAA concentrations are associated with an increased risk of CV mortality in individuals with T2D, independent of clinical and biochemical risk markers.