Objectives Pregnancy is associated with physiological alterations in insulin sensitivity and lipid metabolism. This study investigates the associations between pregestational body mass index (pBMI) and the rate of gestational weight gain (rGWG) in the second trimester with the biomarkers of lipid, fatty acids metabolism and insulin resistance. Methods Sixty nine pregnant women followed. The body weights of the pregnant women were measured and blood samples were obtained at 11–14th and 24–28th weeks of pregnancy. Glucose, total cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, insulin levels and fatty acids were measured. Rate of GWG (kg/week) and The Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) were calculated. The pregnant women were stratified according to their pBMI and the 2nd trimester rGWG. Results The rate of GWG was significantly higher for the group with pBMI<25, compared to the group with pBMI≥25 (p=0.024). Triglyceride, total cholesterol, LDL and HDL cholesterol were significantly increased in the second trimester compared with the first trimester. Palmitic acid, oleic acid, linoleic acid, myristic acid, docosahexaenoic acid (DHA), arachidonic acid (AA), total omega-6 (n − 6) and omega-3 (n − 3) fatty acid levels and n − 6/n − 3 ratio were significantly higher in the second trimester. Glucose was significantly decreased and insulin was increased in the second trimester. In the overweight/obese group; HOMA-IR, insulin, AA, palmitoleic acid and stearic acid were found to be high in comparison to the group with low/normal pBMI. No parameters were associated with rGWG. Conclusions The changes in lipid parameters, free fatty acids, insulin and HOMA-IR in the second trimester were compatible with the changes in lipid metabolism and the development of insulin resistance. Pregestational BMI was shown to have a stronger influence on lipid profile, insulin resistance, and fatty acids than rGWG.