Production of trimethylamine-N-oxide (TMAO), a biomarker of CVD risk, is dependent on intestinal microbiota, but little is known of dietary conditions promoting changes in gut microbial communities. Resistant starches (RS) alter the human microbiota. We sought to determine whether diets varying in RS and carbohydrate (CHO) content affect plasma TMAO levels. We also assessed postprandial glucose and insulin responses and plasma lipid changes to diets high and low in RS. In a cross-over trial, fifty-two men and women consumed a 2-week baseline diet (41 percentage of energy (%E) CHO, 40 % fat, 19 % protein), followed by 2-week high- and low-RS diets separated by 2-week washouts. RS diets were assigned at random within the context of higher (51–53 %E) v. lower CHO (39–40 %E) intake. Measurements were obtained in the fasting state and, for glucose and insulin, during a meal test matching the composition of the assigned diet. With lower CHO intake, plasma TMAO, carnitine, betaine and γ-butyrobetaine concentrations were higher after the high- v. low-RS diet (P<0·01 each). These metabolites were not differentially affected by high v. low RS when CHO intake was high. Although the high-RS meal reduced postprandial insulin and glucose responses when CHO intake was low (P<0·01 each), RS did not affect fasting lipids, lipoproteins, glucose or insulin irrespective of dietary CHO content. In conclusion, a lower-CHO diet high in RS was associated with higher plasma TMAO levels. These findings, together with the absence of change in fasting lipids, suggest that short-term high-RS diets do not improve markers of cardiometabolic health.