N1-methylnicotinamide (N1-MN) and N1-methyl-2-pyridone-5-carboxamide (2Py) are successive end products of NAD+ catabolism. N1-MN excretion in 24-h urine is the established biomarker of niacin nutritional status, and recently shown to be reduced in renal transplant recipients (RTR). However, it is unclear whether 2Py excretion is increased in this population, and, if so, whether a shift in excretion of N1-MN to 2Py can be attributed to kidney function. Hence, we assessed the 24-h urinary excretion of 2Py and N1-MN in RTR and kidney donors before and after kidney donation, and investigated associations of the urinary ratio of 2Py to N1-MN (2Py/N1-MN) with kidney function, and independent determinants of urinary 2Py/N1-MN in RTR. The urinary excretion of 2Py and N1-MN was measured in a cross-sectional cohort of 660 RTR and 275 healthy kidney donors with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Linear regression analyses were used to investigate associations and determinants of urinary 2Py/N1-MN. Median 2Py excretion was 178.1 (130.3-242.8) μmol/day in RTR, compared to 155.6 (119.6-217.6) μmol/day in kidney donors (p < 0.001). In kidney donors, urinary 2Py/N1-MN increased significantly after kidney donation (4.0 ± 1.4 to 5.2 ± 1.5, respectively; p < 0.001). Smoking, alcohol consumption, diabetes, high-density lipoprotein (HDL), high-sensitivity C-reactive protein (hs-CRP) and estimated glomerular filtration rate (eGFR) were identified as independent determinants of urinary 2Py/N1-MN in RTR. In conclusion, the 24-h urinary excretion of 2Py is higher in RTR than in kidney donors, and urinary 2Py/N1-MN increases after kidney donation. As our data furthermore reveal strong associations of urinary 2Py/N1-MN with kidney function, interpretation of both N1-MN and 2Py excretion may be recommended for assessment of niacin nutritional status in conditions of impaired kidney function.