The aim of this study was to evaluate the effects of treating Holstein cows with pegbovigrastim on periparturient diseases, milk production, and reproductive performance while exploring the mode of action of an immunomodulatory protein. Cows were randomly allocated to 1 of 2 treatments, untreated control (CTR, n = 423) and pegbovigrastim (PEG, n = 417). At 7 d from the anticipated calving date (d -7), cows allocated to PEG received a subcutaneous injection of 15 mg of pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim injection, Imrestor, Elanco Animal Health, Greenfield, IN). A second injection was administered within 24 h after calving (d 0). Blood samples were obtained from a subset of cows (CTR, n = 103; PEG, n = 102) at -7 and 0, 3, 7, and 14 d relative to parturition. Samples were used for hemogram and quantification of haptoglobin, nonesterified fatty acids, β-hydroxybutyrate, and trace and macro minerals. Vaginal cytobrush was performed on the same subset cows at d 0, 7, and 14 to assess the relative neutrophil count. Additionally, colostrum samples were collected to measure IgG, IgM, IgA, and lactoferrin concentrations. Postpartum disease occurrence was recorded from calving until 30 d in milk (DIM). Weekly milk yield was recorded for the first 12 wk after calving. Cows treated with PEG had a 3- to 4-fold increase in circulating polymorphonuclear leukocyte, lymphocyte, and monocyte numbers, with a peak at 3 d after treatment followed by a gradual decline, but the counts remained significantly greater compared with CTR at 14 DIM. The administration of PEG did not affect the incidence of clinical and subclinical mastitis, retained fetal membranes, metritis, puerperal metritis, and endometritis. Primiparous cows treated with PEG tended to have lower odds of developing hyperketonemia than CTR [odds ratio (OR) = 0.57, 95% confidence interval (CI) = 0.23 to 1.42]. Cows treated with PEG had higher odds of being diagnosed with lameness within 30 DIM compared with CTR (OR = 1.79, 95% CI = 1.16 to 2.76); however, we found no significant differences by 60 DIM. Treatment with PEG increased the odds of displaced abomasum (OR = 8.27, 95% CI = 1.02 to 66.6). Cows treated with PEG had higher odds of being diagnosed with 1 or more clinical diseases compared with CTR cows (OR = 1.39, 95% CI = 1.02 to 1.90). We observed no differences in linear scores or milk composition between treatments. Furthermore, primiparous cows treated with PEG produced more milk than CTR primiparous cows during the first 12 wk postpartum (PEG = 37.51 ± 0.66; CTR = 35.91 ± 0.65 kg), but no differences were observed on energy-corrected milk. Treatment did not alter reproductive performance; additionally, cows diagnosed with metritis or puerperal metritis and treated with PEG tended to have higher proportion of neutrophils in the vaginal mucosa when compared with CTR metritic cows. Although PEG treatment increased circulating polymorphonuclear leukocyte, monocyte, and lymphocyte numbers, as expected, it was detrimental to cow health because it increased morbidity.