Interleukin-12 (IL-12) is one of cytokines that regulate the innate and adaptive immune responses produced by macrophages. This study was to investigate the effect of Black Cumin Seed Oil (BCSO) to the structure of peripheral blood, phagocytic activity, and secretion of IL-12 by macrophage peritoneal Sprague-Dawley (SD) rat. The 36 female Sprague Dawley (SD) rats divided into 6 groups. Normal group was only given standards food and drink. Treatments group were given BCSO 0.25, 2.5 and 5 ml/kg BW/day for 14 d. Positive control group was given thymoquinone 50 mg/kg BW/day for 14 d. Solvent control group was given DMSO. Blood test, phagocytic activity and secretion of IL-12 by macrophage in vitro performed on the 15th day. Latex and NBT assay method were conducted to measure the phagocytic activity of macrophages. The expression level of IL-12 and TLR4 were analysed by ELISA. Data were statistically tested by one-way ANOVA with significance 95%. BCSO was increase phagocytic activity, secretion of IL-12 by macrophage, and the expression of TLR4. The highest phagocytosis percent (56.83 ± 6.37%) and phagocytosis index (5.18 ± 0.39) was performed by 0.25 BCSO group and did not significantly different with thymoquinone group. The highest level of IL-12 was in 2.5 BCSO group (66.33 ± 2.11 μM).