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 B1 (AFB1) 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 AFB1 at 0 or 40 µg/kg of feed and LAB at 0 or 3 × 1010 cfu/kg of feed, and the LAB was a mixture of equal amounts of Lactobacillus acidophilus, Lactobacillus plantarum, and Enterococcus faecium. The results showed that there were highly significant (P < 0.01) effects of AFB1 toxicity, LAB protection, and their interaction on ADFI, ADG, and G:F of broilers during d 1 to 35. Compared with the AFB1 diet, the LAB diet reduced (P < 0.05) the residues of AFB1 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 (P < 0.05) the contents of aflatoxin M1, a metabolite of AFB1, 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 (P ≤ 0.01) effects of LAB and AFB1 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 (P < 0.05) GSH, GST, and GR by a range of 11.6 to 86.1%, and compared with the AFB1 diet, the LAB diet increased (P < 0.05) GSH, GST, and GR by a range of 24.1 to 146.9%. In the liver, there were interactions (P < 0.05) on GSH and GST on d 14 and on GSH on d 35; in the mucosa, interactions were significant (P ≤ 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 AFB1 by modulating toxin metabolism and activating the GSH pathway in animals.