Heme oxygenase‐1 (HO‐1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF‐7 and T47D) express HO‐1. When overexpressed, we found this enzyme to have anti‐proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1‐MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO‐1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti‐proliferative HO‐1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO‐1. We also describe that HO‐1 inhibits IDO at the post‐translational level through heme starvation. In vivo, we show that rat normal breast expresses HO‐1 and IDO. In contrast, N‐nitrosomethylurea‐induced breast adenocarcinomas only express IDO. In conclusion, we show that HO‐1/IDO cross‐regulation modulates apoptosis and proliferation in rat and human breast cancer cells.—Hill, M., Pereira, V., Chauveau, C., Zagani, R., Remy, S., Tesson, L., Mazal, D., Ubillos, L., Brion, R., Ashgar, K., Mashreghi, M. F., Kotsch, K., Moffett, J., Doebis, C., Seifert, M., Boczkowski, J., Osinaga, E., Anegon, I. Heme oxygenase‐1 inhibits rat and human breast cancer cells proliferation: mutual cross inhibition with indoleamine 2,3‐dioxygenase. FASEB J. 19, 1957–1968 (2005)