Supplementation of OmniGen-AF (OG) improves innate immunity and affects stress hormone concentrations in blood. However, it is not known if immune benefits persist in lactating cows after OG is removed from the diet. The aim was to evaluate the effect of withdrawing OG from the diet on performance, stress hormone concentrations, and peripheral blood mononuclear cells (PBMC) proliferation of mid-lactation dairy cows. Multiparous Holstein cows (n = 32), blocked by parity and days in milk (109 ± 39 d), were randomly assigned to 2 dietary treatments within each block: TMR top dressed with OG (56 g/d) or placebo (CON, 56 g/d). Cows were housed in the same free-stall pen, and fed individually 1×/d through Calan gates. All cows were fed the same diet containing OG before the onset of treatments, for at least 60 d. Cows were milked 3×/d and milk yield was recorded at each milking. Milk composition was analyzed weekly. Body weight and condition score were measured weekly. Blood samples were collected at −1, 1, 3, 5, and 7 wk relative to the onset of treatments for cortisol and prolactin (PRL) analyses, and isolation of PBMC. PBMC were cultured with hydrocortisone, PRL, concanavalin A (ConA), lipopolysaccharides (LPS), or their combinations for 72 h ex vivo to determine proliferative responses. Data were analyzed using PROC MIXED of SAS. Withdrawing OG from the diet of mid-lactation dairy cows with no physical signs of disease and not subjected to an in vivo immune challenge did not affect (P > 0.1) milk yield or composition, DMI, body weight, and blood concentrations of cortisol and PRL. Compared with CON cows, OG cows maintained greater body condition score (P = 0.04). Relative to CON cows, PBMC isolated from OG cows had greater proliferative rate when stimulated by LPS (P = 0.03), but not by ConA ex vivo. There were no interactions (P > 0.1) between dietary treatments and addition of hormones on PBMC proliferation. In conclusion, withdrawing OG from the diet of mid-lactation cows impaired proliferative responses of PBMC stimulated by LPS, suggesting OG supplementation enhances immune response.