Heat stress (HS) is a severe environmental challenge to the dairy industry as it negatively affects milk production. Therefore, it is of great significance to understand the molecular mechanism underlying mammary gland responses to HS. A bovine mammary epithelial cell line, named MAC-T cells, was established from mammary alveolar cells as an in vitro lactation model, where a lactogenic phenotype is induced through hormone supplements. In the present study, multiple culturing conditions were evaluated to differentiate MAC-T cells. The combination of ovine prolactin, dexamethasone, and collagen gel coating was determined to be the most efficient in milk-related gene upregulation. Next, differentiated MAC-T cells were incubated at thermal-neutral condition (37°C) or HS condition (42°C) for 1 hr and recovered (37°C) for 2 or 6 hrs. Based on RNA-sequencing and gene function analyses, differentiation treatment increased the expression of genes related to milk fat synthesis. Under HS, MAC-T cells showed a strong heat shock response and upregulation of Ras protein signal transduction. ZBTB7B and other transcription factors were found to be potential regulators of cellular HS regulation. During recovery periods, protein folding activity was elevated in MAC-T cells. Apoptosis regulation were enriched after 2 hr recovery in MAC- T, while cellular signaling and oxidative stress were enriched after 6 hr recovery. Overall, results from the present study suggest that MAC-T cells have a poor milk protein profile, but are adequate for milk lipid studies. MAC-T cells showed increased cellular stress in protein folding during and post HS. Potential regulators were identified for future gene functional studies.