A new bicyclic macrolide, hamuramicin C (1), was isolated from Streptomyces sp. MBP16, a gut bacterial strain of the wasp Vespa crabro flavofasciata. Its 22-membered macrocyclic lactone structure was determined by NMR and mass spectrometry. The relative configurations of hamuramicin C (1) were assigned by J-based configuration analysis utilizing 1H rotating frame Overhauser effect spectroscopy and heteronuclear long-range coupling NMR spectroscopy. Genomic and bioinformatic analyses of the bacterial strain enabled identification of the type-I polyketide synthase pathway, which employs a trans-acyltransferase system. The absolute configurations of 1 were proposed based on the analysis of the sequences of ketoreductases in the modular gene cluster. Moreover, hamuramicin C (1) demonstrated significant inhibitory activity against diverse human cancer cell lines (HCT116, A549, SNU-638, SK-HEP-1, and MDA-MB-231).