Non-α-tocopherol (i.e. β, γ, and δ-tocopherol) supplements are as important as α-tocopherol with regard to maintaining lactating dairy cow health. However, information on non-α-tocopherol bioaccumulation and its effects on immune cell function is not available. A series of experiments were conducted to investigate the effect of mixed tocopherol oil supplement (Tmix; i.e. α, β, γ, and δ-tocopherol) on body accumulation and immune cell functions of the lactating Holstein dairy cow. Tissue, tissue mitochondria, blood and milk were collected from lactating dairy cows to measure the concentration of all four tocopherol isoforms via Tmix supplementation. In addition, polymorphonuclear leukocyte (PMN) were isolated to investigate the effect of Tmix on its function and immune gene expression. In the first experiment, Tmix increased γ-tocopherol concentrations but did not increase the α-tocopherol concentration in three different types of tissues (i.e. liver, mammary gland and muscle) and liver mitochondria. Within those three different types of tissues, liver showed the highest ability to store tocopherol isoforms (i.e. α- and γ-tocopherol) compared to the other two tissues. Also, amounts of α- and γ-tocopherol were detected in liver mitochondria, but limited amounts were detected in mammary gland mitochondria. In the following experiment, Tmix increased γ-tocopherol concentration in milk and blood as determined via every 8 h and daily measurements. Compared to γ-tocopherol, α-tocopherol showed the highest concentration of the tocopherol isoforms in milk and blood. Limited quantities of β- and δ-tocopherol were detected in milk and blood via Tmix supplementation. In the last experiment, Tmix increased PMN chemotaxis function and did not impair the whole blood respiratory burst response of dairy cows, which might be associated with non-α-tocopherol existing in Tmix. Even though Tmix increased the expression of pro-inflammatory genes in PMN, those are needed during the initial immune activation. Overall, the results of the experiments demonstrated that short-term supplementation with Tmix could compensate γ-tocopherol without altering α-tocopherol in dairy cows. The liver showed the highest capability of accumulating tocopherol isoforms compared to the mammary gland and muscle. In addition, Tmix did not harm immune functions or have any apparent effects on animal health in lactating Holstein dairy cows.