Our aim was to quantify the individual contribution of prepregnancy (PP) fat-free mass (FFM), expressed as [PP-FFM index (PP-FFMI) = FFM (kg)/height (m2)], on markers of glucose homeostasis before and during pregnancy. Body composition was assessed in 43 women before pregnancy using air-displacement plethysmography. Blood was drawn at PP and gestational weeks ∼8 and 30. Relationships between body composition (independent) variables and glucose homeostasis (dependent) variables were assessed using adjusted correlations and simple and multiple linear regression analyses. PP-FFMI was the strongest predictor of plasma insulin concentration [squared partial correlation (Pr2) = 17, P = 0.007] and homeostasis model assessment of insulin resistance (HOMA2-IR) (Pr2 = 16, P = 0.010). At gestation week 30, PP-FFMI and gestational weight gain (GWG) were the strongest predictors of insulin concentration (PP-FFMI: Pr2 = 20, P = 0.010; GWG: Pr2 = 12, P = 0.052) and HOMA2-IR (PP-FFMI: Pr2 = 19, P = 0.012; GWG: Pr2 = 13, P = 0.045). After accounting for PP fat mass index (PP-FMI), PP-FFMI and GWG were independently associated with first-phase insulin response (PP-FFMI: Pr2 = 20, P = 0.009; GWG: Pr2 = 15, P = 0.025) and second-phase insulin response (PP-FFMI: Pr2 = 19, P = 0.011; GWG: Pr2 = 17, P = 0.016). PP-FMI was the strongest predictor of an oral glucose tolerance test‒derived estimated metabolic clearance rate of glucose (PP-FMI: Pr2 = 14, P = 0.037) and estimated insulin sensitivity index (PP-FMI: Pr2 = 13, P = 0.047). PP-FFMI was a predictor of markers of glucose homeostasis before and during pregnancy. Studies assessing the effect of skeletal muscle quality on metabolic regulation during pregnancy are warranted.