Objectives were to determine the effects of acidogenic diets (AD) fed for 21 or 42 d on glucose metabolism and tissue insulin responsiveness. Ninety Holstein cows at 230 d of gestation were randomly assigned to receive diets with −70 or −180 mEq/kg DM for 21 (Short) or 42 d (Long). Cows in Short received a diet with +110 mEq/kg from 233 to 254 d of gestation. Therefore, in the first 21 d cows were fed +110, −70, or −180 mEq/kg DM, whereas in the last 21 d of gestation they were fed either −70 or −180 mEq/kg DM. The glucose tolerance tests (GTT) were performed at 250 and 270 d of gestation by infusing 0.25 g of dextrose/kg BW within 2 min. The following day, cows received 0.1 IU of insulin/kg BW intravenously (insulin challenge, IC). Jugular blood was sampled at min −15, −5, and 0 to establish a baseline, and from 5 to 180 min relative to infusions; concentrations of glucose, NEFA, and insulin were determined and area under the curve (AUC) and change in AUC relative to baseline (rAUC) were calculated. Liver was sampled after the GTT and subcutaneous adipose tissue was sampled after the GTT and IC for RT-qPCR. Data were analyzed by ANOVA with mixed models using the Mixed procedure of SAS. At GTT250, the rAUC for glucose increased (P = 0.04) in cows fed AD (+110 = 264.5, −70 = 285.5, and −180 = 270.4 ± 6.4 mM/min), which is explained by the smaller (P < 0.04) rAUC for insulin in cows fed AD (+110 = 1,420, −70 = 1,079, −180 = 852 ± 208 ng/mL/min). At IC250, cows fed AD tended (P = 0.06) to have increased NEFA (+110 = 0.24, −70 = 0.30, and −180 = 0.33 ± 0.04 mM). At GTT270, cows in Long had greater (P < 0.05) rAUC for glucose than cows in Short (Long = 1,201 vs. Short = 1,110 ± 61 mM/min), which is explained by the smaller (P < 0.04) rAUC for insulin in Long than Short (Long = 969 vs. Short = 1,409 ± 157 ng/mL/min). Hepatic expression of G6PC, PCK1, PCK2, and PC did not differ among treatments after GTT250 and GTT270. Treatments did not affect adipose tissue expression of ATGL, ACC, B2AR, HSL, and PLIN1. Metabolic acidosis induced by AD reduced insulin release in response to a GTT and adipose tissue responsiveness to an IC. Feeding AD for 42 d reduced insulin release in response to GTT.