Cows undergoing negative energy balance often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed at evaluating the impact of a folic acid and vitamin B₁₂ supplement and feed restriction on some immune parameters. Sixteen cows at 45 ± 3 DIM were assigned to 8 blocks of 2 cows each according to their milk production during the previous week, 45 ± 6 kg/d, then within each block, they randomly received weekly intramuscular injections of either saline (C) or 260 mg of folic acid and 10 mg of vitamin B₁₂ (V) for 5 wk. On wk 5, the cows were fed 75% of their ad libitum intake, 24 (±2.5) kg of DM/d, during 4 d. Blood sample samples were taken before the beginning of the experiment, just prior feed restriction and after 3 d of feed restriction to evaluate blood cell population, phagocytosis capacity and oxidative burst of polymorphonuclear leukocyte (PMN). The vitamin supplement did not affect any of the tested variables. Feed restriction reduced (P < 0.05) the percentage of PMN positive for phagocytosis. Accordingly, the percentage of PMN that were positive for oxidative burst after being stimulated with PMA was reduced by feed restriction (P < 0.05). Feed restriction did not affect blood cell population. In conclusion, feed restriction affected the functions of PMN, suggesting that the greater risk of infectious diseases in cow experiencing a negative energy balance is related to impaired immune cell functions.