--- - |2- Our data demonstrated that biogenic nanoselenium (BNS) particles activated Nrf2‐ARE pathway through p38, extracellular signal‐regulated kinase 1/2 (ERK1/2), and protein kinase B (AKT) mediated‐phosphorylation of nuclear factor (erythroid‐derived‐2)‐like 2 (Nrf2) to improve the antioxidant function of intestinal epithelial cells. Abstract As the intestinal epithelium is vulnerable to oxidative stress because of frequent enterocyte renewal and continuous exposure to exogenous agents, it is meaningful to figure out how the epithelial cells exert antioxidant function. We previously synthesized a novel biogenic nanoselenium (BNS) particles and proved that BNS could effectively improve intestinal antioxidative function through activating Nrf2‐ARE pathway. The objective of the present study was to investigate the mechanism by which BNS activate Nrf2‐ARE pathway on the physiological function of intestinal epithelial cells. In the present study, we demonstrated that treatment of IPEC‐J2 cells with BNS particles not only elevated the levels of downstream proteins of nuclear factor (erythroid‐derived‐2)‐like 2 (Nrf2) such as heme oxygenase‐1 and NQO‐1 in a time‐dependent manner which started to weaken at 12 hr after treatment but also significantly activated Nrf2, mitogen‐activated protein kinase (MAPK), and protein kinase B (AKT) pathway in a time‐dependent manner within 24 hr. BNS particles significantly increased the content of phosphorylated‐Nrf2, without evident influence on the level of Kelch‐like ECH‐associated protein 1 (Keap1). Moreover, BNS also induced the activation of p38, extracellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase, and AKT while phosphorylating Nrf2. Using specific protein kinase inhibitors, we found that the Nrf2‐phosphorylating and antioxidative effects of BNS particles were abolished when p38, ERK1/2, and AKT were significantly inhibited. Overall, our data demonstrated that BNS particles activated Nrf2‐ARE pathway through p38, ERK1/2, and AKT mediated‐phosphorylation of Nrf2 to improve the antioxidant function of intestinal epithelial cells - 'Journal of Cellular Physiology, EarlyView. '