Abstract The objectives of this experiment were to compare the circulating concentrations of cortisol and determine whether these correlated with the expression profiles of a set of candidate apoptosis genes in neutrophils of Holstein-Friesian cows milked once vs. 3 times daily for 28 d postpartum. Cows on the once-daily milking regimen had significantly higher plasma cortisol concentrations on d 3, 14, and 28 postpartum than did those milked 3 times daily. On d 3 postpartum, when differences in cortisol and neutrophil counts were highest between the groups, mean mRNA abundance of nuclear factor κB p65 subunit, IκBα, X-linked inhibitor of apoptosis; and heat shock protein 70 were higher in neutrophils of the cows milked once daily than in cows milked 3 times daily. However, no correlations were detected among plasma cortisol concentration, neutrophil count, or neutrophil gene expression in this study. Results suggest that the modest neutrophilia associated with once daily milking of cows immediately postpartum may be related to modifications in the cells’ apoptotic program by factors other than cortisol.