Heat stress and dietary Zn source influence mammary function of lactating dairy cows but their impacts on milk fatty acid (FA) composition and mammary metabolism are largely unknown. The objective of this study was to evaluate milk FA profile and mammary gland gene expression related to energy metabolism of lactating cows fed 2 dietary Zn sources under 2 environments. Multiparous lactating Holstein cows (n = 72) were randomly assigned to 4 treatments with a 2 × 2 factorial arrangement. Treatments included 2 environments: cooled (CL) using fans and misters or non-cooled (NC), and 2 dietary Zn sources: 75 mg/kg of Zn hydroxychloride (IZ) or 35 mg/kg Zn hydroxychloride + 40 mg/kg Zn-Methionine complex (ZC). All cows were cooled for 84 d (temperature-humidity index = 73) and fed respective dietary treatments before the environmental challenge for the remaining 84 d (temperature-humidity index = 78) when NC cows were deprived of cooling. After the environmental challenge, milk samples were collect from 24 cows (6/treatment) at 3, 25, and 53 d to measure FA profile by gas chromatograph. Mammary tissue was collected from 32 cows (8/treatment) at 7 and 56 d for gene expression analysis by RT-qPCR. There were environment × Zn interactions for milk C4:0 and C8:0 (P ≤ 0.02), with ZCNC cows having lower C4:0 and ZCCL cows having lower C8:0. Compared with NC, CL cows had greater C12:0 (P = 0.01), and ZC cows tended (P = 0.06) to have greater C12:0 than IZ. Relative to CL, NC cows had lower de novo (P = 0.02) but greater preformed FA (>C16, P = 0.05), and tended (P = 0.10) to have greater polyunsaturated FA. Compared with CL, mammary tissue from NC cows had greater (P ≤ 0.08) gene expression of phosphofructose kinase-1 and 6-phosphogluconate dehydrogenase, but did not alter gene expression of FA synthase and lipoprotein lipase. In conclusion, dietary Zn source elicits different responses on milk short chain FA profile under different environments; heat stress increases preformed but decreases de novo FA, and may alter glycolysis and pentose pathway of the mammary gland.