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Valanimycin is an azoxy‐containing natural product isolated from the fermentation broth of Streptomyces viridifaciens MG456‐hF10. While the biosynthesis of valanimycin has been partially characterized, how the azoxy group is constructed remains obscure. Herein, the membrane protein VlmO and the putative hydrazine synthetase ForJ from the formycin biosynthetic pathway are demonstrated to catalyze N−N bond formation converting O‐(l‐seryl)‐isobutyl hydroxylamine into N‐(isobutylamino)‐l‐serine. Subsequent installation of the azoxy group is shown to be catalyzed by the non‐heme diiron enzyme VlmB in a reaction in which the N−N single bond in the VlmO/ForJ product is oxidized by four electrons to yield the azoxy group. The catalytic cycle of VlmB appears to begin with a resting μ‐oxo diferric complex in VlmB, as supported by Mössbauer spectroscopy. This study also identifies N‐(isobutylamino)‐d‐serine as an alternative substrate for VlmB leading to two azoxy regioisomers. The reactions catalyzed by the kinase VlmJ and the lyase VlmK during the final steps of valanimycin biosynthesis are established as well. The biosynthesis of valanimycin was thus fully reconstituted in vitro using the enzymes VlmO/ForJ, VlmB, VlmJ and VlmK. Importantly, the VlmB‐catalyzed reaction represents the first example of enzyme‐catalyzed azoxy formation and is expected to proceed by an atypical mechanism. [ABSTRACT FROM AUTHOR]