AbstractCamalexin (3‐thiazol‐2′‐yl‐indole) is the major phytoalexin found in Arabidopsis thaliana. Several key intermediates and corresponding enzymes have been identified in camalexin biosynthesis through mutant screening and biochemical experiments. Camalexin is formed when indole‐3‐acetonitrile (IAN) is catalyzed by the cytochrome P450 monooxygenase CYP71A13. Here, we demonstrate that the Arabidopsis GH3.5 protein, a multifunctional acetyl‐amido synthetase, is involved in camalexin biosynthesis via conjugating indole‐3‐carboxylic acid (ICA) and cysteine (Cys) and regulating camalexin biosynthesis genes. Camalexin levels were increased in the activation‐tagged mutant gh3.5–1D in both Col‐0 and cyp71A13–2 mutant backgrounds after pathogen infection. The recombinant GH3.5 protein catalyzed the conjugation of ICA and Cys to form a possible intermediate indole‐3‐acyl‐cysteinate (ICA(Cys)) in vitro. In support of the in vitro reaction, feeding with ICA and Cys increased camalexin levels in Col‐0 and gh3.5–1D. Dihydrocamalexic acid (DHCA), the precursor of camalexin and the substrate for PAD3, was accumulated in gh3.5–1D/pad3–1, suggesting that ICA(Cys) could be an additional precursor of DHCA for camalexin biosynthesis. Furthermore, expression of the major camalexin biosynthesis genes CYP79B2, CYP71A12, CYP71A13 and PAD3 was strongly induced in gh3.5–1D. Our study suggests that GH3.5 is involved in camalexin biosynthesis through direct catalyzation of the formation of ICA(Cys), and upregulation of the major biosynthetic pathway genes.