Aromatic caninurine formamase (AFMID) is an enzyme involved in tryptophan pathway, metabolizing N-formylkynurenine to kynurenine. AFMID had been found significantly down regulated in clear cell renal cell carcinoma (ccRCC) in both tissue and urine samples. Although ccRCC is characterized by a typical Warburg-like phenotype, mitochondrial dysfunction and elevated fat deposition, it is unknown whether AFMID plays a role in tumorigenesis and the development of ccRCC. In present study, AFMID overexpression had inhibitory effects for ccRCC cells, decreasing the rate of cell proliferation. Quantitative proteomics showed that AFMID overexpression altered cellular signaling pathways involved in cell growth, and cellular metabolism pathways, including lipid metabolism and inositol phosphate metabolism. Further urine proteomic analysis indicated that cellular function dysfunction with AFMID overexpression could be reflected in urine. The activity of predicted up-regulators, DDX58, TREX1, TGFB1, SMARCA4, and TNF in ccRCC cells and urine showed opposing change trends. Potential urinary biomarkers were tentatively discovered and further validated using an independent cohort. Protein panel of APOC3, UMOD and LEAP2 achieved an AUC value of 0.862 for the training cohort, and 0.883 for validation cohort. Present study is of significance in terms of highlighting various aspects of pathway changes associated with AFMID enzymes, discovering potential specific biomarkers for potential patient diagnosis and therapeutic targeting.