(1) Background: Gut microbiota-dependent Trimethylamine-N-oxide (TMAO) has been 5 reported to be strongly linked to renal function and to increased cardiovascular events in the 6 general population and in Chronic Kidney Disease (CKD) patients. Considering the lack of data 7 assessing renal handling of TMAO, we conducted this study to explore renal excretion and 8 mechanisms of accumulation of TMAO during CKD. (2) Methods: We prospectively measured 9 glomerular filtration rate (mGFR) with gold standard methods and plasma concentrations of 10 trimethylamine (TMA), TMAO, choline, betaine and carnitine by LC-MS/MS in 124 controls, CKD 11 and hemodialysis (HD) patients. Renal clearance of each metabolite was assessed in a subgroup of 12 32 patients. (3) Results: Plasma TMAO was inversely correlated with mGFR (r 2 =0.388, p<0.001), 13 confirming elevation of TMAO plasma levels in CKD. TMAO clearances were not significantly 14 different from mGFR, with a mean ± SD TMAO fractional excretion of 105 ± 32 %. This suggests a 15 complete renal excretion of TMAO by glomerular filtration with a negligible participation of 16 tubular secretion or reabsorption, during all stages of CKD. Moreover, TMAO was effectively 17 removed within 4 hours of hemodiafiltration, showing a higher fractional reduction value than that 18 of urea (84.9 ± 6.5 % vs 79.2 ± 5.7 %, p = 0.04). (5) Conclusions: This study reports a strong 19 correlation between plasma TMAO levels and mGFR, in CKD, that can be mainly related to a 20 decrease in TMAO glomerular filtration. Clearance data did not support a significant role for 21 tubular secretion in TMAO renal elimination. 22