Glutathione-related enzymes belong to the protection mechanism of the cells against harmful oxidative damage and chemicals. Glutathione S-transferase (GST) is frequently over-expressed in various cancer cells and is involved in drug resistance. Chlorophyllin is an antioxidant molecule interfering with the GST P1-1 activity. The purpose of this study is to evaluate the short- and long-term protective effects of chlorophyllin as an antioxidant molecule on DNA damage, antioxidant enzyme activities, trace elements, and minerals in chemically induced breast cancer model in vivo. In our study, N-methyl-N-nitrosourea (MNU) was used for inducing breast carcinogenesis in female Sprague-Dawley rats. A total of 36 rats were divided into groups as short term and long term. Each group was divided into four sub-groups as control group received physiological saline solution (n = 3), Chl group (n = 5) received chlorophyllin, MNU group (n = 5) was administered MNU, and Chl + MNU group (n = 5) was treated with both chlorophyllin and MNU. Results illustrated that chlorophyllin had a significant anti-genotoxic effect in the short term, and glutathione-related enzyme activities were protected by chlorophyllin treatment in MNU-induced breast cancer model. Additionally, MNU administration impaired mineral and trace element levels including Na, Mg, K, Fe, Zn, and Co in the liver, kidney, spleen, heart, and tumor tissues; however, adverse effects of MNU were recovered upon chlorophyllin treatment in the indicated tissues of the rats. In conclusion, chlorophyllin can be used as an antioxidant molecule to ameliorate adverse effects of MNU by enhancing antioxidant enzyme activities and regulating trace element and mineral balance in several organs and tumor tissue in the breast cancer model.