Incorporating canola meal (CM) into nursery pig diets has been limited due to the adverse effects of glucosinolates, sulfur-containing glycosides, presented in Brassica family oilseeds. This study investigated the effects of acidifier supplementation in high CM diets on the performance, organ weights, blood profiles, and hindgut microbial composition of nursery pigs. A total of 315 nursery pigs, average 6.0 ± 1.2 kg body weight (BW), were assigned to 1 of 5 dietary treatments with 9 replicates (7 pigs/pen) in a randomized complete block design. Treatments included; a corn-soybean meal (SBM) based diet with 20% CM (positive control, PC) and corn-SBM-based diet with 40% CM without (negative control, NC) or with acidifier (NCA), or encapsulated butyrate (NCB), or both (NCAB). Diets met nutrient requirements of NRC (2012) in 3 phases; phase 1 (common diet; 0 to 1 wk), phase 2 (experimental diets; 1 to 3 wk), and phase 3 (experimental diets; 3 to 6 wk). Pig BW and pen feed consumption were measured by phase to investigate the growth performance of pigs. At the end of each phase, 1 pig per pen was bled and then euthanized for measurement of organ weight. Cecal digesta was collected only at the end of phase 3 for microbial composition analysis. Pigs fed the NCA diets had greater (P < 0.05) final BW and average daily gain during phase 3 and had a tendency of greater (P = 0.084) overall gain-to-feed ratio than pigs fed the NC diet. Pigs fed the NCA diet had a greater (χ2 < 0.05) percentage of non-diarrhea feces than pigs fed the PC diet for phase 2 and the overall period. Pigs fed PC diet had higher serum thyroxine (T4) concentration at the end of phase 3 (P < 0.05) than pigs fed NC diets. The addition of acidifier in NC diet decreased (P < 0.05) spleen weight and tended to decrease (P = 0.064) liver weight. Pigs fed the NCAB diet had a lower (P < 0.05) large intestine weight compared with those fed NC diet. Pigs fed the PC or NCA diets tended to have less (P = 0.06) the relative abundance of OTU Ssd-00188 (Agathobacter rectalis 99.23%) and NCAB diet increased (P < 0.05) the relative abundance of OTU Ssd-00001 (Lactobacillus amylovorus 99.64%) in the cecal microbiota compared with those fed NC diet. Overall, these findings support the incorporation of high CM levels (up to 40%) with acidifier supplementation in nursery pig diets, mitigating the negative implications of dietary glucosinolates and providing benefits for both canola growers and pig farmers in regions with substantial canola production.