Abstract—We have recently cloned a novel molecule that interacts with the angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP). In this study, we tested the hypothesis that ATRAP modulates angiotensin II–induced responses in vascular smooth muscle cells. The results of immunoprecipitation and bioluminescence resonance energy transfer assay demonstrated a direct interaction between ATRAP and AT1R at baseline and showed that angiotensin II enhanced the interaction of these proteins >2-fold. The results of immunofluorescence analysis also demonstrated that >65% of ATRAP constitutively colocalized with an endosome marker. Although only 36% of ATRAP colocalized with AT1R at baseline, angiotensin II enhanced the colocalization of these molecules and made 92% of ATRAP colocalize with AT1R on a quantitative fluorescence analysis. Overexpression of ATRAP by adenoviral transfer decreased the cell surface AT1R number from 4.33 to 2.13 fmol/10⁶ cells at baseline and from 3.04 to 1.26 fmol/10⁶ cells even after removal of angiotensin II. ATRAP also suppressed angiotensin II-mediated increases in c-fos gene transcription and transforming growth factor- production. Furthermore, this suppression was accompanied by inhibition of angiotensin II–induced activation of 5-bromodeoxyuridine incorporation. Finally, ATRAP knockdown by small-interference RNA activated angiotensin II–induced c-fos gene expression, which was effectively inhibited by valsartan, an AT1R-specific antagonist. These results indicate that ATRAP promotes internalization of AT1R and attenuates the angiotensin II–mediated c-fos-transforming growth factor- pathway and proliferative response in vascular smooth muscle cells, suggesting a novel strategy to inhibit vascular fibrosis and remodeling through a novel and specific blockade of AT1R signaling.