Gestational diabetes mellitus (GDM) complicates approximately 3-11% of pregnancies and increases the risk on prenatal morbidity and later development of type 2 diabetes mellitus. Physical activity and sedentary behaviour are thought to play a role in the development of GDM, independent of overweight and obesity. The aim of this study was to examine the relationships between physical activity, sedentary behaviour and the development of GDM using a population-based prospective cohort study. Data from the youngest (1973-1978) cohort of the Australian Longitudinal Study on Women's Health (n=2913) were used to determine the influences of self-reported physical activity, and sedentary behaviour in 2000 and 2003 on the development of GDM over subsequent three year periods, with adjustment for socio-demographic and lifestyle factors. In this cohort of Australian women, physical activity and sedentary behaviour in 2000 and 2003 were not associated with the development of GDM in the subsequent three years. In adjusted models, odds ratios for the development of GDM were 1.92 (95% CI 1.25-2.96) for overweight women (BMI 25-30kg/m2) and 3.11 (1.92-5.03) for obese women (BMI≥30kg/m2) compared with normal weight women. Those with lower education and women born in an Asian country also had higher risk of developing GDM than more highly educated and Australian born women, respectively. In conclusion, pre-pregnancy physical activity and sedentary behaviour appear to be less important in the development of GDM in this cohort than overweight and obesity.