Supplementary folic acid and vitamin B12 have been reported to alter energy partitioning and to increase insulin and glucose plasma concentrations in early lactation. The present study evaluated the effect of these supplements on the response of fed-restricted cows to an intravenous glucose tolerance test (IVGTT). Sixteen cows (45 (SD 3) DIM) were assigned to 8 blocks of 2 cows each according to their milk production during the previous week, 45 (SD 6) kg/d, then within each block, they randomly received weekly intramuscular injections of either saline (C) or 260 mg of folic acid and 10 mg of vitamin B₁₂ (V) for 5 wk. On wk 5, the cows were fed 75% of their ad libitum intake, 24 (SD 2.5) kg of DM/d, during 4 d. On d5, an IVGTT (0.3 g glucose/kg of BW) was administered in 7.5 (SD 1.7) min. Blood samples were taken 20 and 10 min before the infusion (baseline) and 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160 and 180 min post-infusion to measure plasma concentrations of glucose, insulin and nonesterified fatty acids (NEFA). Glucose and insulin positive incremental area under the curve (AUC), peak height, glucose clearance rate at 60 min (CR), NEFA nadir, time to reach the insulin peak or NEFA nadir were compared in a model with treatment as a fixed effect and block as random effect; milk total solid yield the day before the IVGTT was used as a covariable. Insulin AUC tended to be lower (P = 0.07) for V than C, 86 vs. 136 (SE 23) µg.180 min/L. Glucose AUC (274 vs. 252 ± 14 mM.180 min), CR (1.91 vs. 1.82 ± 0.05%/min) and peak height (14.0 vs. 13.5 ± 0.3 mM), insulin peak height (3.2 vs. 4.9 ± 0.84 µg/L), time to reach the peak (6.2 vs. 5.1 ± 0.3 min) and NEFA nadir (162 vs. 164 ± 28 µM) did not differ (P ≥ 0.16) between V and C. NEFA nadir tended to be reached earlier for V than C (P = 0.06; 35 vs. 47 ± 4 min). The reduction in insulin release without changes on intravenous glucose tolerance suggests that the vitamin supplement improved insulin sensitivity in fed-restricted lactating dairy cows.