Dysregulated Raf/MEK/extracellular signal-regulated kinase (ERK) signaling, a common hallmark of tumorigenesis, can trigger innate tumor-suppressive mechanisms, which must be inactivated for carcinogenesis to occur. This innate tumor-suppressive signaling may provide a potential therapeutic target. Here we report that mortalin (HSPA9/GRP75/PBP74) is a novel negative regulator of Raf/MEK/ERK and may provide a target for the reactivation of tumor-suppressive signaling of the pathway in cancer. We found that mortalin is present in the MEK1/MEK2 proteome and is upregulated in human melanoma biopsy specimens. In different MEK/ERK-activated cancer cell lines, mortalin depletion induced cell death and growth arrest, which was accompanied by increased p21(CIP1) transcription and MEK/ERK activity. Remarkably, MEK/ERK activity was necessary for mortalin depletion to induce p21(CIP1) expression in B-Raf(V600E)-transformed cancer cells regardless of their p53 status. In contrast, in cell types exhibiting normal MEK/ERK status, mortalin overexpression suppressed B-Raf(V600E)- or ΔRaf-1:ER-induced MEK/ERK activation, p21(CIP1) expression, and cell cycle arrest. Other HSP70 family chaperones could not effectively replace mortalin for p21(CIP1) regulation, suggesting a unique role for mortalin. These findings reveal a novel mechanism underlying p21(CIP1) regulation in MEK/ERK-activated cancer and identify mortalin as a molecular switch that mediates the tumor-suppressive versus oncogenic result of dysregulated Raf/MEK/ERK signaling. Our study also demonstrates that p21(CIP1) has dual effects under mortalin-depleted conditions, i.e., mediating cell cycle arrest while limiting cell death.