This study aimed to determine the effects of vitamin C supplementation on antioxidant and inflammatory status and cell viability of bovine peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMCs) were isolated from eighteen clinically healthy Japanese Black calves in this study. PBMCs were cultured with vitamin C (vitamin C group) and without vitamin C (control group) and stimulated with or without lipopolysaccharide (LPS). As a result, the total antioxidant capacities, which are the reducing power of components from Fe3⁺ to Fe 2⁺, in the cell culture supernatant with or without LPS stimulation were significantly higher in the vitamin C group than those in the control group (P < 0.05, P < 0.01, respectively). Tumor necrosis factor-alpha in the cell culture supernatant with LPS stimulation was significantly higher in the control group than that in the vitamin C group (P < 0.05). The viability of cells cultured with LPS stimulation was significantly higher in the vitamin C group than that in the control group after 72 h of culture (P < 0.05). These results suggested that vitamin C is related to the antioxidant and anti-inflammatory properties and cell viability of PBMCs obtained from calves.