Macrolactams are macrocyclic compounds characterized by an amide bond within a 20- to 34-membered ring, and often exhibit diverse bioactivities such as antimicrobial, cytotoxic, and antibacterial activities. In the biosynthetic pathway, adenylation enzyme (AD) and proline aminopeptidase (PAP) are core enzymes. AD protect the amine group of the starter unit, while PAP removes this protection after elongation, facilitating macrocyclization. Genome mining of our in-house whole-genome database via BLAST searches identified several hit strains containing a macrolactam biosynthetic gene cluster. Notably, analysis of their biosynthetic gene clusters revealed that a Streptomyces sp. strain possesses an AD domain but lacks a PAP domain, in contrast to other hit strains containing both. Based on these genomic features, the strain was identified as a potential producer of a novel macrolactam. Subsequent ^​1H-¹⁵N-HSQC-TOCSY NMR studies on the crude extract revealed the strain produces α-methyl macrolactam. Following culture and extraction, the planar structure of the compound was elucidated using a combination of 1D and 2D NMR spectroscopy, UV, and mass spectrometry. The structure incorporates an amino acid within the ring unprecedentedly.