Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound L-glutaminyl residue. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.