Although deregulation of MEK/extracellular signal-regulated kinase (ERK) activity is a key feature in cancer, high-magnitude MEK/ERK activity can paradoxically induce growth inhibition. Therefore, additional mechanisms may exist to modulate MEK/ERK activity in favor of tumor cell proliferation. We previously reported that mortalin/HSPA9 can facilitate proliferation of certain KRAS and BRAF tumor cells by modulating MEK/ERK activity. In this study, we demonstrated that mortalin can regulate MEK/ERK activity via protein phosphatase 1α (PP1α). We found that PP1α inhibition increases steady-state levels of phosphorylated MEK1/2 in various tumor cells expressing B-RafV600E or K-RasG12C/D Intriguingly, coimmunoprecipitation and in vitro binding assays revealed that mortalin facilitates PP1α-mediated MEK1/2 dephosphorylation by promoting PP1α-MEK1/2 interaction in an ATP-sensitive manner. The region spanning Val482 to Glu491 in the substrate-binding cavity and the substrate lid of mortalin were necessary for these physical interactions, which is consistent with conventional heat shock protein 70 (HSP70)-client interaction mechanisms. Nevertheless, mortalin depletion did not affect cellular PP1α levels or its regulatory phosphorylation, suggesting a nonconventional role for mortalin in promoting PP1α-MEK1/2 interaction. Of note, PP1α was upregulated in human melanoma and pancreatic cancer biopsy specimens in correlation with mortalin upregulation. PP1α may therefore have a role in tumorigenesis in concert with mortalin, which affects MEK/ERK activity in tumor cells.