Renal transplant recipients (RTR) commonly suffer from vitamin B6 deficiency and its functional consequences add to an association with poor long-term outcome. It is unknown whether niacin status is affected in RTR and, if so, whether this affects clinical outcomes, as vitamin B6 is a cofactor in nicotinamide biosynthesis. We compared 24-h urinary excretion of N1-methylnicotinamide (N1-MN) as a biomarker of niacin status in RTR with that in healthy controls, in relation to dietary intake of tryptophan and niacin as well as vitamin B6 status, and investigated whether niacin status is associated with the risk of premature all-cause mortality in RTR. In a prospective cohort of 660 stable RTR with a median follow-up of 5.4 (4.7-6.1) years and 275 healthy kidney donors, 24-h urinary excretion of N1-MN was measured with liquid chromatography-tandem mass spectrometry LC-MS/MS. Dietary intake was assessed by food frequency questionnaires. Prospective associations of N1-MN excretion with mortality were investigated by Cox regression analyses. Median N1-MN excretion was 22.0 (15.8-31.8) μmol/day in RTR, compared to 41.1 (31.6-57.2) μmol/day in healthy kidney donors (p < 0.001). This difference was independent of dietary intake of tryptophan (1059 ± 271 and 1089 ± 308 mg/day; p = 0.19), niacin (17.9 ± 5.2 and 19.2 ± 6.2 mg/day; p < 0.001), plasma vitamin B6 (29.0 (17.5-49.5), and 42.0 (29.8-60.3) nmol/L; p < 0.001), respectively. N1-MN excretion was inversely associated with the risk of all-cause mortality in RTR (HR 0.57; 95% CI 0.45-0.71; p < 0.001), independent of potential confounders. RTR excrete less N1-MN in 24-h urine than healthy controls, and our data suggest that this difference cannot be attributed to lower dietary intake of tryptophan and niacin, nor vitamin B6 status. Importantly, lower 24-h urinary excretion of N1-MN is independently associated with a higher risk of premature all-cause mortality in RTR.