Sporeamicin A is a 14-membered macrolide antibiotic produced by Saccharopolyspora sp. L53-18 with strong antibacterial activity against Gram-positive bacteria. A new minor component, sporeamicin B, was discovered from the culture filtrates of the sporeamicin A-producing strain. This communication describes the isolation and characterization of sporeamicin B. Fermentation was performed at 28°C for 161 hours in a 250-liter fermenter containing a medium of glucose 3%, corn steep liquor 1%, dry yeast 0.6%, cobalt chloride 0.001%, and FS-antifoam 028 0.04% (pH 7.0). The 200-liter culture broth was filtered, and the filtrate was extracted with ethyl acetate at pH 9.0. Isolation followed the general procedure for basic macrolide antibiotics and was purified by precipitation, silica gel column chromatography, and preparative reverse-phase HPLC (TSK gel 120T, Tosoh). Yield was monitored by bioautography using Micrococcus luteus ATCC9341 and HPLC analysis with a Hitachi gel No. 3056 column (416mm×15cm), UV detection at 275nm, mobile phase CH3CN-MeOH-1/15m AcONH4 (50:25:35), and flow rate 0.8ml/min. Sporeamicin B is basic, soluble in methanol, ethanol, acetone, ethyl acetate, benzene, chloroform, and acidic water, but barely soluble or insoluble in n-hexane and water. It showed positive color reactions with potassium permanganate, iodine, Dragendorff, and Molisch reagents, and negative reactions with ninhydrin and Sakaguchi reagent. Its molecular formula was determined as C36H61NO12 via FAB-MS ((M+H)+, m/z 700) and elemental analysis. The UV spectrum (276nm) indicated an enone function, while the IR spectrum showed enone (1620, 1690cm⁻¹), ester carbonyl (1740cm⁻¹), and hydroxyl (3450cm⁻¹) functions. Structure determination using CI-MS and NMR data revealed that sporeamicin B differs from sporeamicin A by replacing the neutral sugar cladinose with mycarose (supported by CI-MS fragment ions at m/z 158, 381, 556 and the absence of the OCH3 signal at 3.29ppm in ¹H NMR compared to sporeamicin A). Sporeamicin B exhibited antibacterial activity against Gram-positive bacteria, which was approximately twofold less than that of sporeamicin A.