Tripartilactam (1) is a natural macrocyclic lactam originally reported to have a unique [18,8,4]-tricyclic framework. However, the validity of this structure has been contested since niizalactam C (2), bearing a [18,6,6]-tricyclic skeleton, was proposed as an alternative structure in 2015. In the present study, a comprehensive reinvestigation of NMR spectroscopic data and a 13C-13C COSY NMR experiment identified direct 13C-13C coupling, thus leading to the unequivocal revision of the structure of tripartilactam as niizalactam C (2). In addition, whole-genome sequencing analysis of the tripartilactam-producing bacterial strain and subsequent bioinformatics and mutagenesis analyses identified its biosynthetic pathway, which probably utilizes one of the type I polyketide synthase (PKS) modules iteratively during its biosynthesis and exhibits spontaneous [4+2] cycloaddition from the precursor compound, sceliphrolactam, in the post-PKS process.