BACKGROUND: Piperlongumine (PL) is a natural alkaloid obtained from the long pepper and can inhibit the progression of various tumors. However, its role in renal cell carcinoma (RCC) remains unclear. Thus, the purpose of this study was to determine whether PL can suppress RCC progression and to clarify the related mechanisms. METHODS: Cell Counting Kit-8 (CCK-8) and colony formation assays were applied to characterize the effect of PL in RCC cell proliferation; meanwhile, cellular reactive oxygen species (ROS), Fe level, and mitochondrial membrane potential (MMP) assays were used to determine PL's role in mitochondrial homeostasis. Immunofluorescence experiments using MitoTracker red was applied to visualize the morphology of mitochondria. Western blotting and coimmunoprecipitation (Co-IP) and RNA immunoprecipitation (RNA-IP) assays were used to examine PL's regulation of DDX11 expression via miR-15b-3p. In addition, a xenograft mouse model was created to clarify the effect of DDX11 overexpression on RCC progression with or without PL treatment. RESULTS: We found that PL inhibited RCC cell proliferation in a dose-dependent manner by interfering with the mitochondrial homeostasis of RCC cells. In terms of mechanism, RNA sequencing showed that PL decreased the expression of DDX11, inhibited the maturation of miR-15b-3p, and further increased the level of dihydrolipoamide dehydrogenase (DLD) to disrupt the mitochondrial homeostasis of RCC cells. In addition, the vivo xenograft mouse model showed that DDX11 plays a stimulatory role in PL-induced RCC inhibition. CONCLUSIONS: Our study demonstrated that DDX11 contributes to PL-induced RCC inhibition by modulating the miR-15b-3p-DLD axis.