The recruitment of trans-acting enzymes by nonribosomal peptide synthetase (NRPS) assembly line is rarely reported. ColB1 is a flavin-dependent dehydrogenase that is recruited by an NRPS terminal condensation domain (Ct domain) and catalyzes peptidyl carrier protein (PCP)-tethered cysteine dehydrogenation in collismycin biosynthesis. We here report the crystal structure of ColB1 complexed with FAD and reveal a typical structural fold of acyl-CoA dehydrogenases (ACADs). However, ColB1 shows distinct structural features from ACADs in substrate recognition both at the entrance of and inside the active site. Site-directed mutagenesis and substrate modeling establish a Glu393-mediated catalytic mechanism, by which the cysteine substrate is sandwiched between Glu393 and FAD to facilitate Cα proton abstraction and Cβ hydride migration. A ColB1-PCP-Ct complex model is generated, providing structural basis for the unique recruitment interactions between ColB1 and the associated NRPS. These results add insights into the mechanisms by which trans-acting enzymes function in an assembly line.