Abstract Introduction The mechanism by which oral glutamine (GLN) prevents 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer is unknown. While GLN triples the negative extraction of gut glutathione (GSH) in rats, DMBA significantly disrupts it. Actual gut GSH flux has not been reported. We hypothesized that the gut is a producer of GSH; DMBA blocks gut GSH production and supplemental oral GLN antagonizes this effect. Materials and methods Eighty Sprague–Dawley rats were randomized to four groups ( n = 20/group): DMBA + GLN, DMBA + FA, OIL + GLN, OIL + FA. Rats (age 50 days) were gavaged with a one-time dose of 100 mg/kg DMBA or oil. Rats were gavaged with AES-14 as GLN (1 gm/kg/day) or an isonitrogenous amount of Freamine (FA) from 1 week before till sacrifice at 1 week after DMBA (greatest effect on gut GSH extraction). Arterial and portal blood was taken for GLN and GSH levels, and blood flow was measured using 14C-PAH. Gut GLN and GSH fluxes (uptake or production) were calculated. Results DMBA abrogated the normal GSH production (negative flux) in OIL + FA while not affecting GLN metabolism. GLN maintained GSH production in DMBA + GLN. Conclusions Oral GLN restores to normal GSH production in DMBA-treated animals suggesting one of the mechanism(s) by which GLN prevents breast cancer in this model. Unchanged uptake of GLN in the DMBA-treated animals may indicate a block in GSH transport rather than actual intracellular production.