Research supports the beneficial effect of choline on metabolic health and productive performance of transition dairy cows. However, research evaluating the impact of choline on immunity and disease incidence is limited. The objective was to assess the impact of choline on the inflammatory response of stimulated and non-stimulated immune cells (neutrophils [PMN] and mononuclear cells [PBMC]) from 16 Holstein cows during the transition period (7.9 ± 1.7 DIM, n = 8) and mid-lactation (123.6 ± 3.7 DIM, n = 8). Blood immune cells were isolated using density gradient media and were incubated at 37°C and 5% CO₂. First, cells were incubated for 2 (PMN) or 24 h (PBMC) with 1 of 3 supplemental levels of choline (0, 5, or 10 µM). Then PMN were primed or not with LPS (1 µg/mL) for 30 min, followed by a 50-min labeled E. coli phagocytic and oxidative burst assay (included negative controls); PBMC were challenged or not with concanavalin-A (10 µg/mL) for 48 h, followed by a 24-h proliferation assay. Data were transformed to attain normality and analyzed as a randomized block design. Phagocytosis tended to be attenuated by choline if PMN were primed with LPS (79.4 vs. 76.4 ± 4.3% of cells, P = 0.06). Regardless of LPS priming, oxidative burst was attenuated by choline supplementation (61.4 vs. 58.8 ± 7.9%, P = 0.05). The proliferation of PBMC from cows in mid-lactation, but not that of transition cows, was attenuated (P < 0.01) with choline supplementation. These findings suggest that choline can conditionally regulate the inflammatory response of immune cells. Evaluating the expression pattern of genes involved in choline metabolism and inflammation may uncover potential mechanisms of choline action on immune cells.