OBJECTIVE We prospectively evaluated plasma amino acids (AAs) in early pregnancy and midpregnancy and their interplay with phospholipid fatty acids (FAs) in association with gestational diabetes mellitus (GDM) risk. RESEARCH DESIGN AND METHODS From a longitudinal pregnancy cohort of 2,802 individuals, concentrations of 24 plasma AAs at 10–14 and 15–26 gestational weeks (GW) were assessed among 107 GDM case subjects and 214 non-GDM control subjects. We estimated adjusted odds ratios (OR) and 95% CI for the associations of plasma AAs and the joint associations of plasma AAs and phospholipid FAs with GDM risk, adjusting for risk factors including age, prepregnancy BMI, and family history of diabetes. RESULTS Glycine at 10–14 GW was inversely associated with GDM (adjusted OR [95% CI] per SD increment: 0.55 [0.39–0.79]). Alanine, aspartic acid, and glutamic acid at 10–14 GW were positively associated with GDM (1.43 [1.08–1.88], 1.41 [1.11–1.80], and 1.39 [0.98–1.98]). At 15–26 GW, findings for glycine, alanine, aspartic acid, and the glutamine–to–glutamic acid ratio were consistent with the directions observed at 10–14 GW. Isoleucine, phenylalanine, and tyrosine were positively associated with GDM (1.64 [1.19–2.27], 1.15 [0.87–1.53], and 1.56 [1.16–2.09]). All P values for linear trend were <0.05. Several AAs and phospholipid FAs were significantly and jointly associated with GDM. For instance, the lowest risk was observed among women with higher glycine and lower even-chain saturated FAs at 10–14 GW (adjusted OR [95% CI] 0.15 [0.06, 0.37]). CONCLUSIONS Plasma AAs may be implicated in GDM development starting in early pregnancy. Associations of AAs with GDM may be enhanced in the copresence of phospholipid FA profile.