Kidney cancer is a common adult malignancy in the United States. Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, is characterized by widespread metabolic changes. Urea metabolism is one such altered pathway in ccRCC. This is reflected in the lowered mRNA and protein levels of enzymes ASS1, ASL, and ARG2 in tumor samples when compared to the normal kidney. These three enzymes play a critical role in nitrogen metabolism, and alterations in their levels and functions have profound effects on cellular growth. ARG2 is located in the mitochondria of renal cells, and its loss promotes growth by conserving pools of an essential biosynthetic co-factor, pyridoxal -5- phosphate, and preventing a toxic build-up of polyamines. However, ASS1 and ASL are cytosolic in their subcellular location, and ostensibly act independently of ARG2 to suppress growth in ccRCC by depleting cellular aspartate pools and altering pyrimidine synthesis. Overall, our results uncover a delicate metabolic balance in ccRCC cells that is disrupted by the re-expression of urea cycle enzymes. Taken together, our data seek to establish a metabolic tumor suppressive role for the urea cycle enzymes in ccRCC.