Plasma phospholipid fatty acids (FAs) in early and mid-pregnancy have been prospectively related to gestational diabetes mellitus (GDM) risk. Yet, changes of FAs following GDM diagnosis and treatment and their implications for glucose metabolism and control remain understudied. From the Eunice Kennedy Shriver National Institute Child Health and Human Development Fetal Growth Studies-Singleton Cohort of 2802 pregnant women, we ascertained 85 GDM cases using the Carpenter and Coustan criteria and 85 non-GDM controls after exclusion. Using plasma collected before (23-31 weeks) and after GDM diagnosis (33-39 weeks), we quantified 25 saturated, poly- and monounsaturated FAs levels. We estimated the fold change of FAs before and after GDM diagnosis, using multiple linear mixed models adjusting for confounders. Eight FAs showed significant fold changes from the baseline values (23-31 weeks) among GDM cases as compared to women without GDM. Five FAs showed reduced fold changes [myristic acid (14:0): β: -0.22 (95% CI: -0.30, -0.14), palmitic acid (16:0): β: -0.02 (95% CI: -0.04, -0.01), cis-palmitoleic acid (16:1n7): β: -0.15 (95% CI: -0.24, -0.05), alpha-linolenic acid (18:3n3): β: -0.19 (95% CI: -0.31, -0.07], and dihomo-gamma-linoleic acid (20:3n6): β:-0.16; 95% CI: -0.21, -0.11)], whereas 3 showed increases [heptadecanoic acid (17:0): β: 0.17 (95% CI: 0.11, 0.22), cis-vaccenic acid (18:1n7): β: 0.06 (95% CI: 0.03, 0.10), and arachidonic acid (20:4n6): β: 0.10 (95% CI: 0.06, 0.13)]. Our study identified 8 FAs with unique patterns of change before and after GDM diagnosis that differed significantly between women with and without GDM. Our findings may shed light on the role of FA metabolism in the pathophysiology and disease management and progression of GDM. NCT00912132.