BACKGROUND: The metabolic enzyme nicotinamide-N-methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour-promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target. METHODS: NNMT expression was assessed in primary ccRCC (n = 134), non-tumour tissue and ccRCC-derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single-cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT-depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2-deoxy-D-glucose for glycolysis and BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl-sulfide) for glutamine metabolism was investigated in RCC cell lines (786-O, A498) and in two 2D ccRCC-derived primary cultures and three 3D ccRCC air-liquid interface models. RESULTS: NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10 ) and ccRCC-derived metastases (p = 3.92 × 10 ), irrespective of metastatic location, versus non-tumour tissue. Single-cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC-hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5-12.4; KIRC-HR = 3.3, 95% CI: 2.0-5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT-depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2-deoxy-D-glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis-derived models. In two out of three patient-derived ccRCC air-liquid interface models, NNMTi treatment induced cytotoxicity. CONCLUSIONS: Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small-molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.