Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. Here, we identified threonine-265 (Thr265) in ZIPK as a major autophosphorylation site. Mutational analyses revealed that autophosphorylation of Thr265 were essential for its full catalytic activity toward an exogenous substrate as well as for cell death induction. Furthermore, leukemia inhibitory factor (LIF) stimulated Thr265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription (STAT3). Taken together, our findings demonstrate that ZIPK is positively regulated through Thr265 phosphorylation and that this phosphorylation is essential for its function.