Evidence is limited regarding the association of plasma niacin with the risk of hyperlipidemia in participants with diabetes. We aimed to determine the relationship between plasma niacinamide/nicotinic acid and hyperlipidemia in participants with/without diabetes. Plasma niacinamide/nicotinic acid concentrations were measured using high-performance liquid chromatography-tandem mass spectroscopy. Multivariable logistic regression analyses were performed to evaluate the association between plasma niacin and hyperlipidemia in participants with diabetes and non-diabetes in a cross-sectional study. Compared to the first quartile, plasma nicotinamide, nicotinic acid, and niacin (nicotinamide plus nicotinic acid) were associated with a 54%, 50%, and 52% lower risk of hyperlipidemia in diabetic participants, respectively, but no significant association was observed in non-diabetic participants. These inverse associations persisted across subgroups stratified by sex, age, BMI, smoking status, alcohol consumption, and physical activity. In addition, the fully adjusted odds ratios (95% confidence intervals) for hypercholesterolemia and hypertriglyceridemia among diabetic participants were 0.54 (0.38, 0.77) and 0.61 (0.44, 0.85), respectively, when comparing to the first quartile of plasma niacin concentrations (all Ptrend < 0.001). This study of 2647 participants observed that plasma niacin was inversely associated with hyperlipidemia in those with diabetes.