Dimethylbenzanthracene (DMBA) compounds were proven to suppress bone marrow activity, immunosuppressive, and genotoxic and carcinogenic. Black cumin seed extract was used as an immunomodulator, which can be developed as a great therapeutic agent for adjuvant chemotherapy in cancer patients as an immunomodulatory agent. The aim of this study is to determine the effect of black cumin seed hexane extract (BCSHE) as an immunomodulator in SD rat’s peritoneal macrophages induced by DMBA or without DMBA. In vitro experimental study was performed on rat’s peritoneal macrophages cultures induced by DMBA and without DMBA. BCSHE were administered to cultures in five different doses; 1, 5, 25, 125 and 625 μg/ml with control media, tween, and positive control thymoquinone. The test substance was added after the macrophages were activated and then measured by using latex phagocytosis method while ROI secretion assay was measured by using NBT assay method. Results showed that macrophage activation by BCSHE solution increased ROI secretory activity in both SD rat’s macrophage induced by DMBA and without DMBA. BCSHE could increase phagocytic activity in-vitro. It can be concluded that BCSHE can increase ROI secretion and phagocytic activity on SD rat’s peritoneal macrophages induced by DMBA and without DMBA.