Study objectives were to evaluate effects of chronic lipopolysaccharide (LPS) infusion on the acute phase response and immune cell dynamics in lactating cows. Following acclimation (3d), cows (164 ± 22 DIM; 676 ± 16 kg BW; parity 3.1 ± 0.4) were enrolled in a study composed of 2 experimental periods (P); during P1 (3d), cows consumed feed ad-libitum and baseline values were obtained. At the initiation of P2 (7d) cows were assigned to 1 of 2 treatments: 1) saline-infused and pair-fed (CON-PF; 40 mL/h saline; n = 6) or 2) continuous LPS-infused and ad libitum-fed (LPS-AL; E. coli O55:B5; 0.017, 0.020, 0.026, 0.036, 0.055, 0.088, and 0.148 µg/kg BW/h for d 1–7, respectively; n = 6). Blood samples for analysis of acute phase proteins were collected on d 1 and 2 of P1 and 1, 3, 5, and 7 d of P2, while samples for complete blood count analysis were obtained twice daily throughout P2. Effects of treatment, day, and treatment by day interaction were assessed using PROC MIXED (SAS Inst. Inc., Cary, NC). In LPS-infused cows, overall circulating LPS-binding protein (LBP) was increased 40% relative to PF-CON cows (P < 0.05). Relative to baseline, LBP was increased 102% in LPS-AL cows 1 d postbolus and steadily declined with time (P < 0.01). Relative to PF-CON cows, overall SAA concentrations were increased 118% in LPS-AL cows (P < 0.04). Peak SAA concentrations occurred 3 d post-LPS infusion (213% increase relative to baseline; P = 0.01) and gradually returned to baseline with time. Circulating lymphocytes tended to be increased with peak counts at 1.5 d postbolus (32% relative to PF-CON; P < 0.09) after which no treatment differences were observed. In LPS-AL cows, neutrophils initially increased 45% on d 1 (P < 0.03) and then tended to be decreased on d 3.5, 4, and 6 (28, 33, 35%, respectively; P = 0.10) relative to PF-CON cows. Conversely, circulating monocytes were decreased on d 1 and 1.5 d postbolus (57 and 53%, respectively; P = 0.05) and then were increased on d 3, 4, and 6 (71, 69, 69%, respectively; P = 0.05), relative to PF-CON. In summary, initial immunostimulation was attenuated by the development of tolerance to exponentially increasing amounts of LPS.