The objective was to determine if in vitro glucocorticoids treatment of bovine neutrophils would impair their function to phagocytize and kill opsonized M. bovis. We hypothesized that in vitro treatment of bovine neutrophils by glucocorticoids impairs phagocytosis of opsonized M. bovis compared with non-treated neutrophils and such impairment would be a function of M. bovis strain differences. Neutrophils isolated from 20 mid-lactation cows were treated with 5 × 10⁻⁴M dexamethasone (TX) or non-treated (CX). After treatment neutrophil function included: percentage reduction in log₁₀ of M. bovis cfu/mL, percentage of phagocytizing neutrophils, phagocytized M. bovis per neutrophil, and killed M. bovis per neutrophil were quantified by incubating one of TX (NDM1–4) or CX (NM1–4) neutrophils group with one of 4 opsonized M. bovis strains. Least squares means of all neutrophil groups were contrasted using linear mixed–effects models. Overall means ± SEM for the dependent variables of percentage reduction in log₁₀ of M. bovis cfu/mL, percentage phagocytizing neutrophils, phagocytized M. bovis per neutrophil, and killed M. bovis per neutrophil were: 17 ± 1.19, 69 ± 1.48, 8.3 ± 1.23, and 1.57 ± 0.67 for control neutrophils and 14 ± 1.3, 34 ± 1.4, 2.6 ± 0.82, and 0.59 ± 0.53 for neutrophils treated with dexamethasone respectively. Effects due to strain, treatment, and their interaction on neutrophil function measured by the number of phagocytized M. bovis per neutrophil and number of killed M. bovis per neutrophil were different (P < 0.05). However, there was no strain by treatment interaction effect on percentage reduction in log₁₀ of M. bovis cfu/mL or strain and strain by treatment interaction effects associated with the dependent variable of percentage phagocytizing neutrophils. Dexamethasone consistently decreased all neutrophil function tested (P < 0.0001). These findings might explain in part the association of stressful events with subsequent outbreaks of Mycoplasma bovis associated bovine diseases.