When dairy cows are exposed to high-temperature environment, their antioxidant capacity and productive performance decrease, leading to economic losses. Emerging evidence has shown that selenium (Se) can effectively alleviate heat stress in dairy cows; however, the cellular mechanism underlying this protection is not clear. The purpose of this study was to investigate and compare the protective effects of inorganic Se (sodium selenite, SS) and organic Se (selenite methionine, SM) in MAC-T (mammary alveolar cells-large T antigen, a bovine mammary epithelial cell (BMEC) line) cells during heat stress. MAC-T cells were treated in 4 ways unless otherwise described: (i) cells in the heat treatment (HT) group were cultured at 42.5°C for 1 h and then recovered in 37°C for another 12 h; (ii) the SM group was pretreated with organic Se for 2 h, cultured at 42.5°C for 1 h, and then recovered in 37°C for 12 h; (iii) the SS group was treated similarly to the SM group except that the cells were pretreated with inorganic Se instead of organic Se; and (iv) the control group was continuously cultured in 37°C and received no Se treatment. The results showed that heat shock at 42.5°C for 1 h triggered heat shock response, sabotaged the redox balance, and reduced cell viability in MAC-T cells; and pretreatment of cells with SM or SS effectively alleviated the negative effects of heat shock on the cells. However, the cells were much more sensitive to SS treatment but more tolerant to SM. In addition, two forms of Se appeared to affect the expression of different genes, including nuclear factor erythroid 2-related factor 2 (Nrf2) and inducible nitric oxide synthase (iNOS) in the SM group and thioredoxin reductase 1 (TXNRD1) in the SS group in Nrf2-ARE (antioxidant response element) antioxidant pathway and inflammation response. In summary, results showed the mechanistic differences in the protective effects of organic and inorganic Se on heat stress in BMECs.