The objective was to examine effects of environment, dietary Zn source and an intra-mammary lipopolysaccharide (LPS) challenge on plasma concentration of heat shock protein (Hsp) 70, and Hsp gene expression in the mammary gland (MG). Seventy-two multiparous lactating cows were randomly assigned to 4 treatments (n = 18/treatment) in a 2 × 2 factorial arrangement, during summer. Cows were either cooled (CL) or not cooled (NC) and fed diets supplemented with 75 ppm Zn hydroxychloride or 35 ppm Zn hydroxychloride + 40 ppm Zn-Met complex. During the first 84 d of the experiment, all cows were cooled with fans and misters (temperature-humidity index = 73) and fed respective dietary treatments. Starting at 85 d, NC cows were deprived of cooling (temperature-humidity index = 78) for the following 84 d (environmental challenge). Plasma was collected at 0, 12, 40, 68, 82, 85, 87, 89, 96, 110, 125, 138, 152, 165 d of the experiment. A subset of 16 cows was subjected to an intra-mammary LPS challenge at 118 d. Plasma was collected at −4, 0, 3, 6, 12, 24, 48, 96, and 144 h relative to LPS infusion. Plasma concentrations of Hsp70 were analyzed by ELISA. Mammary biopsies were collected from another subset of 30 cows at 0, 91, and 140 d of the trial to analyze gene expression of Hsp90, 70, and 27. Data were analyzed using the mixed procedure of SAS. There were no (P > 0.15) differences due to dietary Zn source or Zn source × environment interaction for any parameters examined. During environmental challenge, NC cows had higher plasma Hsp70 at 165 d and tended to have higher values at 110 and 125 d compared with CL (environment × d, P < 0.01). After LPS challenge, NC cows tended to have higher plasma concentration of Hsp70 at 12 h, but lower at 48 h (environment × h, P < 0.11) than CL. Relative to CL, MG of NC cows had greater (P < 0.05) gene expression of Hsp90, 70, and 27. In conclusion, deprivation of cooling during summer increased gene expression of Hsp of MG and plasma concentration of Hsp70. However, dietary Zn source had no impact on Hsp expression in blood or MG.