Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes. Atg9 is the only integral membrane protein among autophagy-related (Atg) proteins thought to carry the membrane source for forming autophagosomes. Here we show that Drosophila Atg9 interacts with Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2) to regulate the c-Jun N-terminal kinase (JNK) signaling pathway. Significantly, depletion of Atg9 and dTRAF2 compromised JNK-mediated intestinal stem cell proliferation and autophagy induction upon bacterial infection and oxidative stress stimulation. In mammalian cells, mAtg9 interacts with TRAF6, the homolog of dTRAF2, and plays an essential role in regulating oxidative stress-induced JNK activation. Moreover, we found that ROS-induced autophagy acts as a negative feedback regulator of JNK activity by dissociating Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. Our findings indicate a dual role for Atg9 in the regulation of JNK signaling and autophagy under oxidative stress conditions.