Neutrophils are innate immunity cells that represent the first line of cellular defense against invading pathogens. Dairy calves, however, experience neutrophil dysfunction during the first weeks of age, contributing to increased disease susceptibility during this period. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that improved neutrophil function in neonates of other species and mature cows. Nevertheless, its capability to improve neonatal calf neutrophil function is unknown. Therefore, our objective was to evaluate the effect of GM-CSF on the functional capabilities of neonatal calves’ neutrophils in vitro. We hypothesized that supplementation of neonatal neutrophils with GM-CSF would increase microbicidal functions to levels comparable to those of mature immunocompetent cattle. For this, we isolated blood neutrophils from 12 healthy 2-3-d-old Holstein calves and neutrophils from 6 mid-lactation Holstein cows were used as reference of a robust neutrophil function. Subsequently, neutrophils from both calves and cattle were incubated for 9 h with four concentrations (0, 0.005, 0.05, or 0.5 µg/mL) of GM-CSF and microbicidal function of neutrophils was assessed in terms of phagocytosis, respiratory burst, myeloperoxidase (MPO) activity, and extracellular trap formation. Mixed models with Tukey pairwise comparisons were used to identify differences among treatment and age groups. GM-CSF supplementation in vitro increased phagocytosis and MPO activity of calf and cow neutrophils (P<0.001), although not concentration-dependently. Respiratory burst (P=0.644) and extracellular trap formation (P=0.751) were not affected by GM-CSF supplementation. All the microbicidal capacity functions assessed were lower in neutrophils from calves (P≤0.004), but supplementation with GM-CSF increased phagocytosis and MPO activity of calf neutrophils to levels comparable to cow unsupplemented neutrophils. Collectively, our results demonstrated that in vitro supplementation of calf neutrophils with GM-CSF enhanced some functional microbicidal capabilities to levels comparable to immunocompetent cattle. Hence, it may be possible to augment the functional capacity of calf neutrophils in vivo through the therapeutic application of GM-CSF and consequently enhance calves’ resistance to infections. This should be tested in future in vivo studies.