Nrf2-ARE signaling plays a key role in cellular antioxidant-defense system, but whether Nrf2 activation has protective effects against heat shock (HS) stress remains unclear. The objective of the present study was to determine whether the Nrf2 activator, tBHQ, could attenuate the heat stress-induced responses in bovine mammary epithelial cells (bMECs). MAC-T cells were exposed to HS (42.5°C for 1 h) followed by recovery at 37°C to mimic HS stress. Compared with cells consistently cultured at normothermia (37°C), cell viability was dropped after HS treatment (P < 0.01), and arrived at the lowest (64.5 ± 7.68%) after 12 h recovery. Accordingly, mRNA abundance of cell-apoptosis marker genes (Bax and CHOP, P < 0.01) and cellular antioxidant defense genes (HO-1 and Txnrd1, P < 0.01) increased time-dependently in HS challenged cells and reached to the highest level after 12 h recovery, compared with normothermic cultured MAC-T cells. When the MAC-T was pretreated with tBHQ (10 μM) for 2 h and performed HS following 12 h recovery, pre-incubation of tBHQ significantly prevented loss of cell viability and downregulated mRNA expression of Bax and CHOP (P < 0.01) than HS treated MAC-T cells in the absence of tBHQ. The presence of tBHQ also significantly blocked accumulation of reactive oxygen species induced by HS in MAC-T cells after recovering for 2 h (P < 0.01). More importantly, tBHQ pre-treated cells showed stronger activation of Nrf2-ARE signaling compared with the HS group, including more nuclei-accumulations of Nrf2 and higher upregulations of Nrf2-ARE driven gene expressions (including Nrf2, HO-1 and Txnrd1, P < 0.01). RNA silencing of Nrf2 in HS-treated MAC-T cells almost abolished the cytoprotective effects of tBHQ. These results indicated that HS could cause oxidative stress in bMECs, and stabilization of Nrf2 by tBHQ could attenuate HS-induced bMECs damage.