A novel β-methyl macrolactam, designated YAK07.353, was isolated  from Kitasatospora sp. YAK07, discovered in soil at the College of Pharmacy, Seoul National University. Using two primers targeting adenylation enzyme and proline aminopeptidase involved in macrolactam biosynthesis, strain YAK07 was screened and yielded a new compound with a molecular weight of 353. YAK07.353 was found to be highly labile to protic solvents, necessitating minimal contact with water or methanol. To overcome this instability, the compound was fractionated by normal-phase chromatography (EtOAc:MeOH 5:1), followed by reverse-phase HPLC. After purification, 1D- and 2D-NMR analyses enabled the elucidation of its planar structure, revealing a β-methyl macrolactam scaffold bearing two hydroxyl groups.