We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)‐anchor biosynthesis display enhanced TGF‐β responses. These studies implicated the involvement of a 150 kDa GPI‐anchored TGF‐β1 binding protein, r150, in modulating TGF‐β signaling. Here, we sought to determine the molecular identity of r150 by affinity purification and microsequencing. Our results identify r150 as CD109, a novel member of the α2‐macroglob‐ulin (α2M)/complement superfamily, whose function has remained obscure. In addition, we have identified a novel CD109 isoform that occurs in the human placenta but not keratinocytes. Biochemical studies show that r150 contains an internal thioester bond, a defining feature of the α2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF‐β receptor system, and a negative modulator of TGF‐β responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF‐β signaling independently of ligand sequestration and may exert its effect on TGF‐β signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF‐β signaling suggest that CD109 plays a unique role in the regulation of isoform‐specific TGF‐β signaling in keratinocytes.—Finnson, K. W., Tam, B. Y. Y., Liu, K., Marcoux, A., Lepage, P., Roy, S., Bizet, A. A., Philip, A. Identification of CD109 as a TGF‐β1 accessory receptor in human keratinocytes. FASEB J. 20, E780–E795 (2006)