Recently, new macrolide antibiotics possessing various biological activities have been isolated from fermentation broths of streptomycetes. In the course of our search for antimicrobial substances from microorganisms, a new antibacterial antibiotic, X-14952B (1) was isolated from the fermentation broth of a Streptomyces sp. We report the structure elucidation of 1 by means of 1H and 13C NMR spectroscopic analyses. Antibiotic 1 has a molecular formula C42H69NO12 deduced from FAB-MS, elemental analysis and 13C NMR. The 13C NMR spectrum demonstrated that 1 is structurally similar to irumamycin (2), with the presence of 3-O-carbamoyl-2-deoxy-β-D-rhamnoside indicated by anomeric and carbamoyl carbons, and the aglycone moiety coinciding with 2. However, 1 lacks the epoxy carbons of 2 but has additional oxygen-bonded and methine carbons, indicating a different alkyl side chain. The structure of the C12 alkyl side chain of 1 was deduced from 2D-NMR analysis and a retroaldol reaction which yielded 3-hexanone. Combined evidence supports structure 1 for X-14952B as a 20-membered macrolide lactone attached to a neutral sugar and a C12 alkyl sidechain. Notably, the slight structural difference in the side-chains of 1 and irumamycin (2) results in 1 being primarily an antibacterial agent and 2 an antifungal agent, similar to structure-activity effects seen in other macrolides like concanamycins, virustomycin A, bafilomycin and L-681,110A.