Obesity during pregnancy may be correlated with an adverse nutritional status affecting pregnancy and offspring outcomes. We examined the associations of prepregnancy body mass index and gestational weight gain with plasma fatty acid concentrations in mid-pregnancy. This study was embedded in a population-based prospective cohort study among 5636 women. We obtained prepregnancy body mass index and maximum weight gain during pregnancy by questionnaires. We measured concentrations of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n-3 polyunsaturated fatty acid (n-3 PUFA) and n-6 polyunsaturated fatty acid (n-6 PUFA) at a median gestational age of 20.5 (95 % range 17.1-24.9) weeks. We used multivariate linear regression models. As compared to normal weight women, obese women had higher total SFA concentrations difference: 0.10 standard deviation (SD) (95 % Confidence Interval (CI) 0, 0.19) and lower total n-3 PUFA concentrations difference: - 0.11 SD (95 % CI - 0.20, - 0.02). As compared to women with sufficient gestational weight gain, those with excessive gestational weight gain had higher SFA concentrations difference: 0.16 SD (95 % CI 0.08, 0.25), MUFA concentrations difference: 0.16 SD (95 % CI 0.08, 0.24) and n-6 PUFA concentrations difference: 0.12 SD (95 % CI 0.04, 0.21). These results were not materially affected by adjustment for maternal characteristics. Our results suggest that obesity and excessive weight gain during pregnancy are associated with an adverse fatty acids profile. Further studies are needed to assess causality and direction of the observed associations.