Heat stress decreases milk production, compromises animal health, and increases mortality. Study objectives were to understand the biological adaptation of mammary tissue to heat stress via analysis of the transcriptome using RNA-seq. Four multiparous Holstein dairy cows (101 ± 10 DIM; 574 ± 36 kg BW, 38 ± 2 kg milk/d) were randomly assigned to 1 of 4 environment chambers with a crossover design. Following a 9d adaptation period, cows were either subjected to heat stress [HS: 36°C during the day and 32°C during the night; THI = 87.2 and 81.8] for 9d or kept in thermal neutral conditions [20°C; THI = 65.5] for 9d, but pair-fed (PF) with heat-stressed cows. There was a 30d washout period between periods. Mammary biopsies were obtained at the end of each period. HS decreased milk yield (17%) and protein content (4.1%). HiSeq2000 platform was used to measure the mRNA profile. Data were normalized by Lowess prior ANOVA analysis using JMP Genomic (SAS systems) with treatment as the main effect and cow as the random effect. HS had a minor effect on the transcriptome with only 198 differentially expressed genes (DEG; FDR < 0.05; 1.4% of annotated genes measured): 53 upregulated and 145 downregulated genes in HS vs. PF. No gene known to be related to milk or protein synthesis was affected by treatment. Functional analysis was performed using the Dynamic Impact Approach and Database for Annotation, Visualization and Integrated Discovery revealed general inhibition of pathways in HS vs. PF, in particular, pathways affected were protein export, proteasome, basal transcription factors, and steroid biosynthesis. The inhibition of basal transcription factors and protein export may partly explain the observed decrease in milk and protein synthesis. The biological significance of inhibited mammary steroid biosynthesis remains unclear; however, decreased steroidogenesis has been previously reported in heat-stressed bovine granulosa cells. In summary, transcriptome analysis revealed that HS inhibited metabolic activity by decreasing transcripts associated with protein export that might affect lactation performance.