Reactive oxygen species (ROS)-induced activation of Apoptosis signal-regulating kinase 1 (ASK1) plays crucial roles in oxidative stress-mediated cell death through the activation of the JNK and p38 MAPK pathways. However, the regulatory mechanism of ASK1 in the oxidative stress response remains to be elucidated. Here, we identified the kelch repeat protein, Slim, as an activator of ASK1 through a Drosophila misexpression screen. We also performed a proteomics screen and revealed that Kelch domain containing 10 (KLHDC10), a mammalian ortholog of Slim, interacted with Protein phosphatase 5 (PP5), which has been shown to inactivate ASK1 in response to ROS. KLHDC10 bound to the phosphatase domain of PP5 and suppressed its phosphatase activity. Moreover, KLHDC10 was required for H(2)O(2)-induced sustained activation of ASK1 and cell death in Neuro2A cells. These findings suggest that Slim/KLHDC10 is an activator of ASK1, contributing to oxidative stress-induced cell death through the suppression of PP5.