Significance While understanding metabolic change of tumor cells is crucial to improve cancer therapy, current 2D cell culture condition may not fully recapitulate in vivo metabolic environment of tumors. Our metabolomics analysis demonstrates that proline metabolism is critical for the KSHV-transformed cell growth in 3D culture, not in 2D culture. Specifically, the KSHV K1 oncoprotein activates PYCR proline biosynthesis enzyme, increasing intracellular proline concentration for tumor cell growth in in vitro 3D spheroid culture and in vivo tumorigenesis. This study describes an oncogenic strategy of KSHV to enhance proline synthesis for virus-induced transformation, adding the proline metabolic pathway as a potential target for KS treatment.