Polymorphonuclear leukocytes (PMNL) are key cellular components of the immune system and represent the first line of defense against pathogens or other stressors, especially during the periparturient period. The objective was to examine the role of methionine supply during the transition period on PMNL function and mRNA abundance of genes associated with inflammation, oxidative stress, and metabolism. Multiparous Holstein cows were used in a block design and assigned to a control diet or the control plus rumen-protected methionine (MET; Mepron, Evonik Nutrition & Care GmbH, Germany). Mepron was fed from −28 to 30 d relative to parturition at a rate of 0.09% and 0.10% of the dry matter intake during the prepartum and postpartum period, respectively. That rate ensured a ratio of Lys to Met in the metabolizable protein close to 2.8:1. Blood for PMNL extraction and in vitro oxidative burst and phagocytosis was collected at −10, +10, and +21 d relative to parturition. Data were analyzed by using the mixed procedure of SAS considering block as random effect and treatment, time and its interaction as fixed effect. Although the glucose transporter SLC2A1 did not differ, there was an overall upregulation of lactate dehydrogenase in response to feeding MET suggesting increased metabolic activity, which agrees with the greater overall phagocytosis and oxidative burst in those cows. That idea is supported by the upregulation of various components of the Toll-like receptor cascade and both the pro-inflammatory (TLR2, TLR4, NFKB1, and IL1B), and anti-inflammatory response (IL10). Despite greater overall plasma concentration of myeloperoxidase, a microbicidal enzyme, in MET cows, no differences were detected for genes associated with oxidative stress response (MPO, NOX1, SOD1, SOD1, and NOS2). However, expression of genes associated with glutathione synthesis and peroxidase activity were upregulated (GPX1 and GSR) in the MET cows. Overall, the data suggest that enhanced supply of methionine improves immune function of PMNL at least in part through molecular mechanisms associated with energy metabolism, glutathione metabolism, and inflammatory responses.