Rin1, the prototype of a new family of multidomain Rab5 exchange factors, has been shown to play an important role in the endocytosis of the epidermal growth factor receptor (EGFR). Herein, we examined the role of Rin1 in the down-regulation of EGFR following EGF stimulation. We observed that overexpression of Rin1 accelerates EGFR degradation in EGF-stimulated cells. In concordance, depletion of endogenous Rin1 by RNA interference resulted in a substantial reduction of EGFR degradation. We showed that Rin1 interacts with signal-transducing adaptor molecule 2 (STAM2), a protein that associates with hepatocyte growth factor-regulated substrate and plays a key role in the endosomal sorting machinery. Green fluorescent protein (GFP)-Rin1 co-localizes with hemagglutinin (HA)-STAM2 and with endogenous hepatocyte growth factor-regulated substrate. Furthermore, wild type STAM2, but not a deletion mutant lacking the SH3 domain, co-immunoprecipitates with endogenous Rin1. This interaction is dependent on the proline-rich domain (PRD) of Rin1 as Rin1ΔPRD, a mutant lacking the PRD, does not interact with STAM2. Moreover, EGFR degradation was not accelerated by expression of the Rin1ΔPRD mutant. Together these results suggest that Rin1 regulates EGFR degradation in cooperation with STAM, defining a novel role for Rin1 in regulating endosomal trafficking.