Carnosine has been shown to reduce oxidation and glycation of low density lipoprotein hence improving dyslipidaemia in rodents. The effect of carnosine on human plasma lipidome has thus far not been investigated. We aimed to determine whether carnosine supplementation improves the plasma lipidome in overweight and obese individuals. Lipid analysis was performed by liquid chromatography mass spectrometry in 24 overweight and obese adults: 13 were randomly assigned to 2 g carnosine daily and 11 to placebo, and treated for 12 weeks. Carnosine supplementation maintained trihexosylceramide (0.01 ± 0.19 vs −0.28 ± 0.34 nmol/ml, p = 0.04), phosphatidylcholine (77 ± 167 vs −81 ± 196 nmol/ml, p = 0.01) and free cholesterol (20 ± 80 vs −69 ± 80 nmol/ml, p = 0.006) levels compared to placebo. Trihexosylceramide was inversely related with fasting insulin (r = −0.6, p = 0.002), insulin resistance (r = −0.6, p = 0.003), insulin secretion (r = −0.4, p = 0.05) and serum carnosinase 1 activity (r = −0.3, p = 0.05). Both phosphatidylcholine and free cholesterol did not correlate with any cardiometabolic parameters. Our data suggest that carnosine may have beneficial effects on the plasma lipidome. Future larger clinical trials are needed to confirm this.