Lactic acid bacteria (LAB) and the glutathione (GSH) pathway are protective against aflatoxin, but information on the effect of LAB on aflatoxin metabolism and GSH activity in farm animals is scarce. This study aimed to investigate the effects of LAB and aflatoxin B (AFB) on growth performance, aflatoxin metabolism, and GSH pathway activity using 480 male Arbor Acres broiler chickens from d 1 to 35 of age. Diets were arranged in a 2 × 2 factorial design, including AFB at 0 or 40 µg/kg of feed and LAB at 0 or 3 × 10 cfu/kg of feed, and the LAB was a mixture of equal amounts of , , and . The results showed that there were highly significant ( < 0.01) effects of AFB toxicity, LAB protection, and their interaction on ADFI, ADG, and G:F of broilers during d 1 to 35. Compared with the AFB diet, the LAB diet reduced ( < 0.05) the residues of AFB in the liver, kidney, serum, ileal digesta, and excreta on d 14 by 121.5, 80.6, 43.7, 47.0, and 26.5%, respectively, and on d 35 by 40.6, 60.2, 131.7, 37.9, and 32.9%, respectively, whereas the LAB diet increased ( < 0.05) the contents of aflatoxin M, a metabolite of AFB, in the liver, kidney, serum, and ileal digesta on d 14 by 98.2, 154.2, 168.6, 19.1, and 34.1%, respectively, and in the kidney and serum on d 35 by 32.6 and 142.2%, respectively. For the activity of the GSH pathway in the liver and duodenal mucosa, there were significant ( ≤ 0.01) effects of LAB and AFB on reduced GSH, glutathione S-transferases (GST), and glutathione reductase (GR) on d 14 and 35; compared with the control diet, the LAB diet increased ( < 0.05) GSH, GST, and GR by a range of 11.6 to 86.1%, and compared with the AFB diet, the LAB diet increased ( < 0.05) GSH, GST, and GR by a range of 24.1 to 146.9%. In the liver, there were interactions ( < 0.05) on GSH and GST on d 14 and on GSH on d 35; in the mucosa, interactions were significant ( ≤ 0.01) on GSH and GR on d 14 and on GST on d 35. It can be concluded that LAB is effective in the detoxification of AFB by modulating toxin metabolism and activating the GSH pathway in animals.