Abnormal lipid profiles have been associated with gestational diabetes mellitus (GDM), but studies with longitudinal measures of lipids throughout pregnancy are sparse. The aim of the present study was to characterize longitudinal changes in lipid profiles throughout pregnancy and prospectively examine the associations of plasma lipid concentrations with risk of GDM. This study was a nested case-control study including 107 GDM cases and 214 matched non-GDM controls from participants in the National Institute of Child Health and Human Development (NICHD) Fetal Growth Studies – Singleton cohort. Blood samples were collected longitudinally at Gestational Weeks (GW) 10-14, 15-26 (fasting sample), 23-31, and 33-39. Plasma concentrations of triglycerides, total cholesterol, and high-density lipoprotein cholesterol (HDL-C) were measured by enzymatic assays. Low-density lipoprotein cholesterol (LDL-C) was calculated using Friedewald’s formula. Plasma triglycerides, total cholesterol, and LDL-C increased as pregnancy progressed. At GW 10-14, the adjusted odds ratios (aORs) of GDM comparing the highest versus lowest quartile were 3.15 (95% confidence interval [CI] 1.38-7.15; P trend = 0.002) for triglycerides and 0.44 (95% CI 0.18-1.09; P trend = 0.045) for HDL-C. At GW 15-26, the aORs were 6.57 (95% CI 2.25-19.17; P trend = 0.001) for triglycerides and 0.23 (95% CI 0.08-0.63; P trend = 0.005) for HDL-C. No significant associations were observed for total cholesterol and LDL-C concentrations with risk of GDM. Higher plasma triglyceride and lower HDL-C concentrations in early and mid-pregnancy were significantly associated with a greater risk of GDM. Total cholesterol and LDL-C concentrations during pregnancy were not significantly associated with GDM risk.