Previous research in various species has shown that granulocyte-colony stimulating factor stimulates the production and release of neutrophils cells from bone marrow. The objective of this study was to characterize the effects of polyethylene glycol-bound bovine granulocyte colony-stimulating factor [pegbovigrastim (PEG); Imrestor, Elanco] on circulating leukocyte counts. Thirty-four Holstein cows were randomly assigned to receive 2 injections of either physiologic saline (n = 16) or PEG (n = 18), 7 d before expected calving (d −7) and within 24 h after calving (d 0). Cows were sampled at d −7, d −6, d 0, d +1, d +7, and d +21, relative to calving. We included only cows for which the interval from the first injection to calving was ≥3 d, such that the interval (mean ± SD) from first treatment to calving was 6.7 ± 1.9 d. Treatment effects were assessed with mixed linear regression models. After the first injection, neutrophil counts (×10⁹/L) in PEG-treated cows increased from 4.3 (95% CI 3.8 to 4.8) at d −7 to 18.2 (CI 16.3 to 20.3) at d −6 (P < 0.0001). Their counts then decreased from d −6 to d 0, when the second injection was administered, at a rate of −0.31 × 10⁹ neutrophils/L per day (P < 0.0001). After the second injection, neutrophil counts increased from 16.4 (CI 13.7 to 19.6) at d 0 to 32.8 (CI 25.2 to 42.7) at d +1 (P < 0.0001), after which counts decreased at a rate of −3.73 × 10⁹ neutrophils/L per day until d +7 (P < 0.0001). Counts continued to decrease from d +7 to d +21 at a slower rate of −0.43 × 10⁹ neutrophils/L per day (P < 0.0001), until baseline levels were reached. Conversely, in control cows, neutrophil counts were unchanged from d −7 to d −6 (P = 0.86) after the first injection and then decreased from 6.1 (CI 5.0–7.3) at d 0, to 3.2 (CI 2.4–4.2) at d +1 (P < 0.0001) after the second injection. Neutrophil count was greater (P < 0.001) in PEG than control at d −6, 0, +1, and +7. Area under the curve (cells ×10⁹/L per 28 d) for neutrophil counts in the PEG group was 429 versus 99 in the control group (P < 0.0001). The sustained increase in circulating neutrophil count around the time of calving may contribute to improved health during the peripartum transition period.