Treatment of transition cows with granulocyte colony stimulating factor (G-CSF) has been shown to increase neutrophil count and function. It was hypothesized that prepartum under-nutrition may reduce the effect of a commercial recombinant bovine G-CSF product (pegbovigrastim; IMR). Hence this study was undertaken to test the effect of under-nutrition and IMR treatment on gene expression in neutrophils. Pasture-fed cows (n = 99) in New Zealand were blocked by calving date and BCS and randomly assigned in a 2 × 2 factorial design to be fed prepartum to exceed energy requirements or restricted to 85% of energy requirements. Half of the animals in each group were injected with IMR or saline at approximately 7 d before expected calving and again on the day of calving. Blood samples were collected 7 d pre-calving (D-7) and samples from blood, uterus and milk were on D4 and D7 after calving. Gene expression analysis was performed for 21 genes using Nanostring. Effects of time and IMR treatment were observed but feeding did not affect gene expression. On average, cows showed higher expression of almost all selected genes at D4 compared with D-7 including genes for migration and inflammation markers (L-selectin, ICAM-1 and TLR 2 and 4; P < 0.05) indicating an ongoing neutrophil response to the hormonal and metabolic stresses of the parturition and postpartum infections. IMR treatment enhanced the effect by further increasing expression of ICAM1 and TLR2 (P < 0.05) suggesting increased neutrophil efficiency. In uterine fluid and to a lesser degree in milk IMR lowered expression of migration markers and increased expression of genes for other neutrophil functions, including myeloperoxidase, FAS, and caspases 2 and 9 (P < 0.05) potentially increasing neutrophil effectiveness. IMR treatment resulted in significant increases in the expression of genes involved in inflammation, phagocytosis, respiratory burst, degranulation, and apoptosis/survival of neutrophils in blood, uterine fluid and milk, and also migration of blood neutrophils.