BK polyomavirus (BKPyV) is a widespread human DNA virus that presents a significant threat to kidney transplant recipients, leading to polyomavirus-associated nephropathy (PVAN) in 1–10% of cases and resulting in graft loss in approximately 50% of affected individuals. As no effective antivirals exist, treatment relies on reducing immunosuppression, increasing the risk of graft rejection. Therefore, identifying host-directed antiviral strategies is urgently needed.   We have established advanced 3D human kidney organoid models to study BKPyV infection, including adult stem cell-derived kidney tubuloids (KTs) and KTs-on-a-chip, that recapitulate the cellular architecture and complexity of the renal epithelium. BKPyV efficiently infected and spread throughout the nephron epithelial cells in both systems, and infection markedly decreased KT growth rates.   From a previous genome-wide CRISPR/Cas9 knockout screen in renal proximal tubule epithelial cells, we identified the enzyme Methionine Adenosyltransferase 2A (MAT2A) as a critical proviral factor. Pharmacological inhibition of MAT2A significantly reduced BKPyV replication in both 2D cultures and 3D models, delaying expression of early (LTAg) and late (VP1) viral proteins, inhibiting virus production. Furthermore, combination treatment with MAT2A inhibitors and the CFTR inhibitor glibenclamide, which is known to prevent viral entry, demonstrated a synergistic antiviral effect in kidney tubuloids and KTs-on-a-chip.   Together, our findings identify MAT2A as a promising host target for therapeutic intervention against BKPyV.  Moreover, these results establish kidney organoid models as powerful tools for studying BKPyV pathogenesis and for preclinical evaluation of antiviral strategies, contributing to the development of effective host-directed therapies.