The objective of this study was to evaluate cows’ metabolic responses when fed 2 dietary Zn sources under 2 environmental conditions and subjected to an intramammary lipopolysaccharide challenge (IM-LPS). Multiparous lactating Holstein cows (n = 72) were randomly assigned to 4 treatments with a 2 × 2 factorial arrangement. Treatments included 2 different environments (E): cooled (CL) using fans and misters or non-cooled (NC), and 2 dietary Zn sources: 75 ppm Zn hydroxychloride (IZ) or 35 ppm Zn hydroxychloride + 40 ppm Zn-Met complex (ZC). All cows were cooled and fed respective dietary treatments during first 84 d of the trial (baseline phase [BP]). During the following 84 d (environmental challenge [EC]), NC cows were deprived of cooling. Air temperature and relative humidity were recorded and temperature-humidity index was calculated, which averaged 73 and 78 during BP and EC. Plasma was collected at d −73, −45, −31, −17, −3, −1, 1, 3, 5, 12, 26, 41, 54, 68, and 81 relative to EC. At d 34 of EC, a subset of cows (n = 16) were subjected to IM-LPS, with plasma collected at 0, 3, 6, 12, 24, 48, 72, 96, 120, and 144 h after infusion. During BP, ZC cows tended (P = 0.11) to have higher plasma triglyceride than IZ (5.8 vs. 5.5 mg/dL). After EC, a tendency (P = 0.10) for E × diet interaction for plasma glucose was observed, as IZCL had highest. Interactions of E × d (P ≤ 0.06) were observed for β-hydroxybutyrate, triglyceride, nonesterified fatty acids (NEFA) and insulin due to lower metabolites but higher insulin for NC than CL 3 to 5 d after EC. After IM-LPS, ZCNC cows had the lowest NEFA (E × diet, P = 0.05). Relative to IZ, ZC cows tended (P = 0.08) to have higher β-hydroxybutyrate (5.1 vs. 6.0 mg/dL) and had higher (P = 0.04) insulin (0.5 vs. 0.8 µg/L). NC cows had lower (P = 0.05) plasma glucose and higher insulin at 3 and 12 h after IM-LPS (E × h, P < 0.01) than CL. Relative to CL, NC cows had higher plasma NEFA at 3 h but lower at 24, 48 and 120 h of IM-LPS (E × h, P = 0.01). In conclusion, heat stress and dietary Zn source altered lactating cows’ metabolic responses.