OBJECTIVE: Dietary protein is an important modulator of glucose metabolism. However, studies regarding the association between dietary protein intake and gestational diabetes mellitus (GDM) risk are sparse. This study was to examine the association. RESEARCH DESIGN AND METHODS: Our study included 21,457 singleton pregnancies reported among 15,294 participants of the Nurses' Health Study II cohort between 1991 and 2001. Included pregnancies were free of chronic diseases before pregnancy or previous GDM. Generalized estimating equations were used to estimate the relative risks (RRs) and 95% CIs. RESULTS: After adjustment for age, parity, nondietary and dietary factors, and BMI, multivariable RRs (95% CIs) comparing the highest with lowest quintiles were 1.49 (1.03-2.17) for animal protein intake and 0.69 (0.50-0.97) for vegetable protein intake. The substitution of 5% energy from vegetable protein for animal protein was associated with a 51% lower risk of GDM (RR [95% CI], 0.49 [0.29-0.84]). For major dietary protein sources, multivariable RRs (95% CIs) comparing the highest with the lowest quintiles were 2.05 (1.55-2.73) for total red meat and 0.73 (0.56-0.95) for nuts, respectively. The substitution of red meat with poultry, fish, nuts, or legumes showed a significantly lower risk of GDM. CONCLUSIONS: Higher intake of animal protein, in particular red meat, was significantly associated with a greater risk of GDM. By contrast, higher intake of vegetable protein, specifically nuts, was associated with a significantly lower risk. Substitution of vegetable protein for animal protein, as well as substitution of some healthy protein sources for red meat, was associated with a lower risk of GDM.