Gestational diabetes mellitus (GDM) is a growing health concern that typically appears during the latter half of pregnancy. It is defined as any form of diabetes or glucose intolerance with the onset of or first recognition during pregnancy. Pregnant women with GDM are at risk for certain neonatal complications. Our aim was to investigate the association between the serine/threonine kinase 33 (STK33) rs4929949 polymorphism and the risk of diabetes developed during pregnancy in Saudi women. We genotyped the rs4929949 polymorphism in a hospital-based case-control study, comparing pregnant 200 GDM women with 300 non-GDM women. Genotyping was performed using the TaqMan assay method.  All the glucose values and lipid profiles examined, excluding LDL-C, were associated with an increased risk of GDM (p < 0.05). A significant variation with respect to the genotypic and allele distribution in the disease group was observed when compared to the non-GDM women (OR = 1.83 [95% CI = 1.0, 3.1], p = 0.02) and (OR = 1.33 [95% CI = 1.0, 1.7], p = 0.02). The present study revealed a significant association of the STK33 gene polymorphism with GDM women in a Saudi population.