Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3), also known as lysyl hydroxylase 3 (LH3) has been demonstrated to be overexpressed in several kinds of cancers and facilitate cell migration. Currently, we aimed to reveal the role of PLOD3 in renal cell carcinoma (RCC) progression, and explore whether TWIST1 (Twist family bHLH transcription factor 1) is involved in this process. Fifty-eight paired RCC tissues and normal tissues were collected and subjected to qPCR and immunohistochemistry (I11C) technology to detect the expression levels of PLOD3. The clinical value of PLOD3 in predicting RCC progression was then explored. Cell-Counting Kit-8 (CCK-8), wound healing, transwell chambers and tumor-bearing experiments were applied to monitor cell proliferation, migration, invasion and tumorigenesis. Protein levels were determined by using western blotting technology to assess cell apoptosis and epithelial to mesenchymal transition (EMT). PLOD3 expression was enhanced in RCC tissues and cells, which predicted higher T (tumor), N (lymph node) and M (metastasis) stages, histological grade and TNM (tumor, lymph node, metastasis) stage. PLOD3 downregulation in RCC A498 cells obviously inhibited cell proliferation, migration, invasion, EMT and tumorigenesis and increased cell apoptosis. PLOD3 overexpression led to opposite results in RCC A704 cells. PLOD3 downregulation reduced the expression levels of TWIST1, beta-catenin and p-AKT. In addition, TWIST1 overexpression rescued the repressions of cell proliferation, migration, invasion, EMT and the activation of beta-catenin and AKT signaling in addition to apoptosis promotion induced by PLOD3 downregulation. Collectively, this study illustrated that PLOD3 knockdown suppressed RCC malignance via inhibiting TWIST1-mediated activation of beta-catenin and AKT signaling.