Vasopressin (VP) activates protein kinase A (PKA), resulting in phosphorylation events and membrane accumulation of aquaporin-2 (AQP2). Epidermal growth factor receptor (EGFR) inhibition with erlotinib also induces AQP2 membrane trafficking with a phosphorylation pattern similar to VP, but without increasing PKA activity. Here, we identify the ribosomal s6 kinase (RSK) as a major mediator phosphorylating AQP2 in this novel, erlotinib-induced pathway. We found that RSK was expressed in collecting duct principal cells in rat kidneys. RSK inhibition with BI-D1870 blocked erlotinib-induced AQP2 serine 256 (S256) phosphorylation and membrane accumulation. CRISPR-generated RSK knockout (KO) cells failed to show increased S256 phosphorylation in response to erlotinib. Like PKA, RSK was able to phosphorylate AQP2 S256 in vitro. Inhibition of phosphoinositide-dependent kinase-1 (PDK1), a known activator of RSK, blocked erlotinib-induced AQP2 S256 phosphorylation and membrane accumulation. We conclude that RSK is a crucial terminal kinase phosphorylating AQP2 at S256 upon EGFR inhibition by erlotinib.