Serum creatinine and/or creatinine clearance (C_Cr) are routinely used for estimation of glomerular filtration rate as marker of kidney function in humans and also in experimental animal models. Due to renal tubular secretion of creatinine, these parameters often inadequately describe renal function. The exact knowledge of the molecular entities responsible for creatinine secretion is of prominent importance for an appropriate interpretation of serum creatinine and C_Cr in clinical practice and in translational medicine. Here creatinine clearance and the ability of kidneys to accumulate creatinine were measured in wildtype (WT) and organic cation transporters 1 and 2 double knockout (OCT1/2-/-) mice in vivo. The interaction of creatinine with organic cations transport has been also studied in freshly isolated proximal tubules from these mice and also from humans. A significant lower C_Cr and also accumulation of exogenous creatinine was observed in OCT1/2-/- compared with WT mice. Furthermore, creatinine interacts with the transport of organic cations in proximal tubules from WT mice and also from humans, but not in those from OCT1/2-/- mice. Moreover, hOCT2, like also hOCT3, hOAT2 and hOAT3 but not hOAT1, critically mediate the accumulation of creatinine in vitro. In conclusion, OCT2 plays a decisive role in the secretion of creatinine both in human and mouse kidney. This should be taken into account when using creatinine to evaluate renal function in rodents and also under particular situations in humans.