Ovarian cancer (OC) is the most lethal gynecological carcinoma due to the lack of diagnostic markers and effective drug targets. Discovery of new therapeutic targets in OC to improve the treatment outcome is urgently needed. We performed proteomic analysis of OC specimens and the paired normal tissues and revealed that proteins associated with mitochondrial proteostasis and protein translation were highly expressed in ovarian tumor tissues, indicating that mitochondria are required for tumor progression of OC. Heat shock protein 60 (HSP60), an important mitochondrial chaperone, was upregulated in ovarian tumors. HSP60 silencing significantly attenuated growth of OC cells in both cells and mice xenografts. Proteomic analysis revealed that HSP60 silencing downregulated proteins involved in mitochondrial functions and protein synthesis. Metabolomic analysis revealed that HSP60 silencing resulted in a more than 100-fold increase in cellular adenine levels, leading to increased adenosine monophosphate and an activated AMPK pathway, and consequently reduced mTORC1-mediated S6K and 4EBP1 phosphorylation to inhibit protein synthesis that suppressed the proliferation of OC cells. These results suggest that HSP60 knockdown breaks mitochondrial proteostasis, and inactivates the mTOR pathway to inhibit OC progression, suggesting that HSP60 is a potential therapeutic target for OC treatment.