Dairy cows experience a nutrient deficit early postpartum when they also exhibit immune dysfunction and inflammation. Previous work raised a possible association with the reduced activation of the nutrient-sensing AKT/mTOR (mTOR) pathway, but responsiveness of the pathway to pro-inflammatory stimulus was not investigated. The objective was to describe differences in activation (expressed as phosphorylation) of AKT kinase and mTOR substrates 4EBP1 and S6RP in immune cell subsets and their responsiveness to LPS during the transition period. Heparinized blood of 14 Holstein cows were taken on d −38 ± 12, −12 ± 4, 7 ± 2, 21 ± 2, and 41 ± 3 relative to calving. Samples were split and pre-treated with PBS or Amino Acid/Glucose solution (AA) (1 h at 37°C) before stimulation with 100 ng/mL LPS or vehicle for 1 h (treatments: PBS, AA, PBS+LPS, AA+LPS). Ratios of phosphorylated to total AKT, 4EBP1, and S6RP were measured by phospho-flow cytometry after fixing and permeabilizing cells. Cell populations were gated based on morphology (neutrophils [PMN], mononuclear cells [MNC]) and cell surface marker staining (CD14⁺ monocytes). For analysis, PROC MIXED (SAS v. 9.4) with fixed effects of time, treatment, time × treatment, repeated effect of time, and Tukey’s test for multiple comparisons was used. Across all time points, PMN had 2.5, 2.9, and 2.2 x higher phospho-ratio of AKT, 4EBP1, and S6RP compared with MNC (P < 0.01), and 1.8, 2.6, and 1.3 × higher phospho-ratio for the same proteins compared with CD14⁺ (P < 0.01). Ratios were higher for CD14+ compared with all MNC for AKT and S6RP (P < 0.01), but not for 4EBP1 (P = 0.13). Expressed as change from baseline (PBS), both time and treatments had an effect on activation, but no interaction was found (P > 0.08), indicating activation with LPS was independent of time point. Phosphorylation of mTOR substrates reached the maximum increase from baseline when whole blood was pretreated with AA and then stimulated with LPS. In conclusion, ex vivo activation of the mTOR pathway is highest in PMN, stimulation with LPS reliably induced activation of pathway proteins, responsiveness of target proteins was maintained postpartum, and activation is potentiated by supplying nutrients to immune cells.