C3-dominance by immunofluorescence is a defining feature in the diagnosis of C3 glomerulopathy. Most pathologists stain for C3c, which has been reported as a trace/negative even in otherwise clear-cut cases of dense deposit disease. We investigated the usefulness of C3d immunohistochemistry in biopsies with C3 glomerulopathy as an ancillary diagnostic tool. All biopsies from patients diagnosed with C3 glomerulopathy in the period January 2005 to June 2017 in the Erasmus MC, Rotterdam were included (n = 14; 10 C3 glomerulonephritis, 4 dense deposit disease). The staining pattern of C3d and C4d by immunohistochemistry was analyzed. As controls, biopsies from patients with immune complex membranoproliferative glomerulonephritis (n = 2), infection-associated glomerulonephritis (n = 6), pauci-immune crescentic glomerulonephritis (n = 7), tubulointerstitial nephritis (n = 7) and chronic-active antibody-mediated rejection (n = 9) were included. All 14 biopsies with C3 glomerulopathy showed a C3d score of ≥2, including two clear-cut biopsies with C3 glomerulopathy originally showing a trace/negative staining for C3c. In the control group, a C3d score ≥2 was observed in 11 biopsies (35%; 2 with immune complex membranoproliferative glomerulonephritis (100%), 6 with infection-associated glomerulonephritis (100%), 1 with pauci-immune crescentic glomerulonephritis (14%), 1 with tubulointerstitial nephritis (14%) and 1 with chronic-active antibody-mediated rejection (11%)). C4d was positive in 71% of the biopsies with C3 glomerulopathy (10/14). In conclusion, C3d immunohistochemistry is a valuable tool in the diagnosis of C3 glomerulopathy, especially in cases in which C3c immunofluorescence shows a trace/negative. We recommend the use of C3d in addition to C3c in cases suspicious for C3 glomerulopathy.