A large body of evidence supports the hypothesis that proteasomal degradation of the growth suppressor p27(Kip1) (p27) facilitates mammalian cell cycle progression. However, very few studies have addressed the possibility of proteasome-independent mechanisms of p27 proteolysis. Here we provide evidence for a novel pathway of p27 degradation via the lysosome that is mediated by its interaction with the endosomal protein sorting nexin 6 (SNX6), a member of the sorting nexin family of vesicular trafficking regulators. p27 and SNX6 interact in vitro and in vivo in mammalian cells, partially colocalize in endosomes, and are present in purified endosomal fractions. Gain- and loss-of-function studies revealed that SNX6 induces endosomal accumulation of p27. Moreover, p27 is detected in lysosomes and inhibition of lysosome-dependent proteolysis impairs serum-mediated down-regulation of p27 in a SNX6-dependent manner. To validate the localization of p27 in these organelles, we analyzed several cell lines using two different anti-p27 antibodies, several organelle-specific markers [e.g., early endosome antigen 1, lysosomal-associated membrane protein (LAMP) 1, LAMP2, and LysoTracker], and overexpression of fluorescent p27 and SNX6. Remarkably, silencing of SNX6 attenuates p27 down-regulation in the G(1) phase of the mitotic cell cycle and delays cell cycle progression. We therefore conclude that, in addition to the proteasome-dependent pathway, SNX6-mediated endolysosomal degradation of p27 also contributes to cell cycle progression in mammalian cells.