Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor prognosis. Reprogramming of amino acid metabolism is one of the characteristics of PDAC, in which arginine metabolism is significantly altered in PDAC cells and is involved in important signaling pathways. Current studies have identified arginine deprivation as a potential strategy for PDAC treatment. In this study, we performed Liquid Chromatograph Mass Spectrometer (LC-MS)-based non-targeted metabolomic analysis on PDAC cell lines with stable Rio Kinase 3 (RIOK3) knockdown and PDAC tissues with different RIOK3 expressions and found that RIOK3 expression was significantly correlated with arginine metabolism in PDAC. Subsequent RNA sequencing (RNA-Seq) and Western blot analysis showed that RIOK3 knockdown significantly inhibited the expression of arginine transporter solute carrier family 7 member 2 (SLC7A2). Further studies revealed that RIOK3 promoted arginine uptake, mechanistic target of rapamycin complex 1 (mTORC1) activation, cell invasion, and metastasis in PDAC cells via SLC7A2. Finally, we found that patients with high expression of both RIOK3 and infiltrating Treg cells had a worse prognosis. Overall, our study found that RIOK3 in PDAC cells promotes arginine uptake and mTORC1 activation through upregulation of SLC7A2 expression, and also provides a new therapeutic target for therapeutic strategies targeting arginine metabolism.