The aim of this study was to assess whether the levels of physical activity before and during early pregnancy are associated with the prevalence of gestational diabetes mellitus (GDM). The study group included 160 puerperas. Among them, 40 (25%) diagnosed as having GDM during their recent pregnancy, whereas the remaining 120 (75%) served as controls. The international physical activity questionnaire (IPAQ-Greek version) was applied twice, in an attempt to estimate the level of physical activity before and during early pregnancy. Women who were "inactive" before or during early pregnancy had odds ratio (OR) 7.9 [95% confidence interval (CI) 3.7-16.56] and 1.3 (95% CI 1.2-1.4) of developing GDM, compared to "minimally active" or "active" women, respectively. Pregnancy resulted in a decrease in the level of physical activity (P < 0.005) during early pregnancy, independently of the diagnosis of GDM and morbidity during early pregnancy. We conclude that physical inactivity before and during early pregnancy is associated with increased risk for developing GDM in late pregnancy.