A Mediterranean-style eating pattern (MED-EP) may include moderate red meat intake. However, it is unknown if the pro-atherogenic metabolite trimethylamine N-oxide (TMAO) is affected by the amount of red meat consumed with a MED-EP. The results presented are from a secondary, retrospective objective of an investigator-blinded, randomized, crossover, controlled feeding trial (two 5-wk interventions separated by a 4-wk washout) to determine if a MED-EP with 200g unprocessed lean red meat/wk (MED-CONTROL) reduces circulating TMAO concentrations compared to a MED-EP with 500g unprocessed lean red meat/wk (MED-RED). Participants were 27 women and 12 men (n=39 total) who were either overweight or obese (BMI: 30.5 ± 0.3 kg/m2 mean ± SEM). Serum samples were obtained following an overnight fast both before (pre) and after (post) each intervention. Fasting serum TMAO, choline, carnitine, and betaine concentrations were measured using a targeted Liquid chromatography-mass spectrometry. Data were analyzed to assess if (a) TMAO and related metabolites differed by intervention, and (b) if changes in TMAO were associated with changes in Framingham 10-year risk score. Serum TMAO was lower post-intervention following MED-CONTROL compared to MED-RED intervention (post-MED-CONTROL 3.1 ± 0.2 µM vs. post-MED-RED 5.0 ± 0.5 µM, p<0.001), and decreased following MED-CONTROL (pre- vs post-MED-CONTROL, p = 0.025). Exploratory analysis using mixed model analysis of covariance identified a positive association between changes in TMAO and changes in HOMA-IR (p = 0.036). These results suggest that lower amounts of red meat intake leads to lower TMAO concentrations in the context of a MED-EP.