We recently characterized an interaction between the Staphylococcus aureus immune evasion molecule Staphylococcus aureus binder of Ig (Sbi) and complement C3, an interaction mediated primarily through the binding of C3d(g) to Sbi domain IV. Events related to these studies prompted us to investigate via mutagenesis the binding interface of C3d for Sbi domain IV (Sbi-IV), as well as to revisit the controversial issue of the complement receptor 2 (CR2) binding site of C3d. Specifically, we had shown that Sbi domains III and IV fragment binding to C3dg inhibited the latter's binding to CR2. Moreover, a published cocrystal structure of C3d bound to complement inhibitory C-terminal domain of extracellular fibrinogen-binding protein (Efb-C), a structural and functional homolog of Sbi-IV, showed Efb-C binding to a region on the concave face of C3d previously implicated in CR2 binding by our mutagenesis data but not confirmed in the CR2(short consensus repeat [SCR]1-2):C3d cocrystal structure. We have now analyzed by surface plasmon resonance the binding of a series of variant C3dg molecules to biosensor-bound Sbi-IV or CR2(SCR1-2). We found that mutations to the concave face acidic pocket of C3d significantly affected binding to both Sbi-IV and CR2, although there was divergence in which residues were most important in each case. By contrast, no binding defects were seen for mutations made to the sideface of C3d implicated from the cocrystal structure to be involved in binding CR2(SCR1-2). The results with Sbi-IV suggest a mode of binding highly similar to that visualized in the Efb-C:C3d complex. The results with CR2 confirm our earlier mapping studies and cast even further doubt on the physiologic relevance of the complex visualized in the C3d:CR2 cocrystal. The Journal of Immunology, 2010, 184: 1946-1955.