The objective of this study was to determine the effect of starch content in the close-up diet on insulin sensitivity of female calves early in life. Thirty-eight female Holstein heifer calves were born to dams fed either a high (26% starch; HI; n = 20) or moderate (14% starch; MOD; n = 18) starch close-up diet commencing at 28 d before expected calving date. Following birth, calves were removed from the dam within 2 h, and fed 3 2-L meals of colostrum within the first 24 h of life. Calves were housed individually and offered 10 L/d of milk replacer (26% CP, 18% fat mixed to 130 g/L) fed through a Calf Rail feeding system. There was no difference in birth body weight of calves between HI or MOD. A glucose tolerance test (GTT) was performed a minimum of 6 h after their last colostrum, or milk meal on d 2, d 10 ± 2 and d 20 ± 2. The GTT involved an intravenous infusion of glucose at a dose of 180 mg/kg BW via a jugular catheter, with sequential blood sampling for 90 min after the infusion, and samples were analyzed for plasma glucose and insulin concentrations. Data were analyzed using the FIT model of JMP and included the fixed effects of dam treatment, parity and their interaction. There was no difference in basal concentrations of glucose before the GTT at d 2, 10 or 20, and no difference in basal insulin at d 2 or 10, however, at d 20, HI calves had increased basal concentrations of insulin as compared with MOD (3.04 vs. 1.92 ng/mL; P = 0.05). On d 2, HI calves had greater maximum insulin concentrations (11.1 vs. 6.55 ng/mL; P = 0.02) and greater area under the curve for insulin (17.2 vs. 10.8 ng/mL × min; P = 0.03) following the glucose infusion, but during the GTT, with no difference in glucose response. On d 10 HI calves had reduced insulin sensitivity (8.51 vs. 14.3 mg/min × ng/mL; P = 0.03) and tended to have reduced glucose clearance rates, and on d 20, HI calves tended to have higher maximum insulin concentration following the glucose infusion as compared with MOD. These findings suggest that feeding a HI close-up diet may reduce insulin sensitivity of female offspring early in life.