Climate change adversely affects the dairy economy as high temperatures and humidity (i.e., heat stress) result in greater incidence of cattle disease and mortality and lower milk yield. Previous research by our group demonstrated consequences of dry period heat stress on cow health and milk production in the subsequent lactation. However, the molecular mechanisms through which dry period heat stress impairs lactation performance have yet to be fully elucidated. In this study, we evaluated the impacts of dry period heat stress on the mammary proteome across the subsequent lactation. During the dry period (~46 d), multiparous Holstein cows were housed in either shaded barns with fans and water soakers (cooled group [CL]; n = 4) or shaded barns without cooling (heat stressed group [HT]; n = 4) at the University of Florida Dairy Unit. All cows were cooled postpartum. Mammary biopsies were obtained at 14, 42, and 84 DIM. Proteomes were examined using iTRAQ technology at the UF Institute of Biotechnology Research by a Q Exactive Plus mass spectrometry system coupled to the NanoEasy nLC-1200. Data were searched against Uniprot Bos taurus database (45,234 contigs) using ProteinPilot v4.5. A meta-analysis from Student’s t-test was employed to test for protein expression differences between HT and CL at each time point. Proteins were differentially expressed (DEPs) at a fold change of less than 0.7 and greater than 1.3 and a P-value of < 0.05. A total of 4,964 proteins were identified. 251 unique proteins were differentially expressed between HT and CL. There were more upregulated proteins in HT compared with CL and the most DEPs occurred at 84 DIM. DEPs are involved in functions such as the heat shock response (e.g., ST13, HSPB1), energy metabolism (e.g., NDUFA4, ATP5O), carbohydrate metabolism (e.g., MDH1, TALDO1), amino acid metabolism (e.g., IVD, ASRGL1), fatty acid biosynthesis (e.g., ACSF3, ACACA), lactose synthesis (e.g., B4GALT1), and translation (e.g., RPS2, EIF1AX). These processes are critical for milk synthesis and the response to stress and may explain, at least in part, the impaired lactation performance of HT cows.