Immune dysfunction is associated with nutrient deficit in postpartum dairy cows, but the mechanistic link is unclear. The nutrient-sensing mechanistic target of rapamycin (mTOR) signaling pathway is a known regulator of the immune response in other species and our work shows that it is impaired in postpartum dairy cows. Dendritic cells regulate key innate and adaptive immune mechanisms, therefore our objective was to investigate the effect of pharmacological mTOR inhibition in bovine monocyte derived dendritic cells (moDC). Monocytes from 8 cows were differentiated in vitro in the presence or absence of mTOR inhibitors (rapamycin [RAPA], 100 nM). On d 7 of culture inhibitors were removed and cells were stimulated with Lipopolysaccharide (LPS, 100 ng/mL) or left as unstimulated controls. Gene expression of IL-10, TNF-α, IL-12A and IL-12B was measured after 4h. Expression of surface markers CD14, MHCII, CD40 and CD80 was measured after 24h by flow cytometry. All analysis was done using Friedman’s ANOVA, and multiple comparisons corrected with Dunn’s procedure. All cows showed an upregulation of surface MHCII, CD40 and CD80 and a downregulation of CD14 in moDC compared with monocyte populations, indicating a mature phenotype. RAPA differentiated cells had a 2.2-fold higher baseline expression of surface MHCII (P = 0.001), and a 1.8-fold increased expression of surface CD80 (P < 0.001) in response to LPS compared with controls. Stimulation with LPS lead to upregulation of TNF-α, IL-10, IL-12A and IL-12B gene expression both control and RAPA (P < 0.03). Inhibition of mTOR during differentiation induced higher gene expression of TNF-α, IL-12A and IL-12B (P < 0.002) and lower gene expression of IL-10 (P = 0.002) in response to LPS compared with moDC differentiated without inhibitor. Our study shows that inhibition of the mTOR pathway can alter phenotype and cytokine gene expression in bovine moDC in vitro, indicating a more pro-inflammatory profile. An altered inflammatory response of these key regulators could contribute to the immune dysfunction in postpartum dairy cows.