Cells capacity to counteract oxidative stress is controlled by Nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor and a key regulator of antioxidant and phase II detoxification genes. Nrf2 activity is subject to proteosomal degradation promoted by Kelch-like ECH-associated protein 1 (Keap1), but small molecules like tBHQ can antagonize this interaction, enabling Nrf2 activation. Previous studies have demonstrated the neuroprotective effects of Nrf2 activation in nearby astrocytes through the use of pharmacological activators or via over-expression(1,2). However, the extent to which Nrf2 in astrocytes could respond to endogenous signals to mediate neuroprotective responses was unclear. Recently our group has demonstrated that mild oxidative stress and brief ischemic episodes activate endogenous Nrf2 in astrocytes, leading to neuroprotection (3) . In this study we investigate the mechanism underlying the activation of astrocytic Nrf2 by mild oxidative stress in an in vitromodel of primary murine cortical mixed neuron/astrocyte cultures. Contrary to the established dogma, here we will report a novel mechanism through which mild oxidative stress triggers astrocytic Nrf2 activation in a manner, which is distinct from classical Keap1 antagonism via small molecular inhibitors like tBHQ. The mechanism involves direct regulation of Nrf2’s transactivation properties, and can act additively to the classical tBHQ-induced pathway, suggesting that therapeutic manipulation of Nrf2 activity may be achievable even in astrocytes suffering oxidative stress.