Fatty acids (FAs) have been implicated in the development of gestational diabetes mellitus (GDM), but the role of monounsaturated FAs (MUFAs) remains understudied. We investigated the associations of plasma phospholipid MUFAs in early to mid-pregnancy with cardiometabolic biomarkers and GDM risk. From the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Fetal Growth Studies-Singletons cohort (2009-2013), we identified 107 women with GDM according to Carpenter and Coustan criteria and 214 control participants without GDM matched (2:1) on age, race/ethnicity, and gestational week (GW) of blood collection. MUFAs were measured at 10-14, 15-26, 23-31, and 33-39 GWs by gas chromatography mass spectrometry. We found that the concentration of total 18:1 MUFAs was significantly lower among women with GDM than those without GDM at 15-26 GWs. Each SD increment in the level of total 18:1 MUFAs was associated with a 40% lower risk of GDM at 15-26 GWs. Moreover, each SD increment in vaccenic acid (18:1n-7) levels at 10-14 and 15-26 GWs were associated with a 36% and 45% lower risk of GDM, respectively. Our extensive assessments of MUFAs advance our understanding of the unique associations of FA composition with GDM risk, suggesting the potentially beneficial role of MUFAs in GDM pathophysiology.