In the course of a screening program for antimycoplasmal antibiotics of actinomycete origin, Actinomadura sp. No. SA-4427 isolated from a soil sample in Nichinan City, Miyazaki Prefecture, Japan, was found to produce 2'-amino-2'-deoxyadenosine with selective antimycoplasmal activity. The strain was taxonomically classified into Actinomadura (Lechevaliers' Group III B) based on morphological traits (curled spore chains with warty cylindrical spores, colorless/orange vegetative mycelia, white/shell pink aerial mycelia, apricot soluble pigment), cell wall chemistry (positive for meso-diaminopimelic acid), and whole-cell sugar analysis (positive for madurose). Fermentation was performed in seed medium (1% glucose, 2% starch, 0.5% yeast extract, 0.5% peptone, 0.4% CaCO₃) and production medium (2% dextrin, 0.2% glucose, 1.5% soybean meal, 0.3% yeast extract, 0.3% CaCO₃) at 27°C. The antibiotic was isolated via Amberlite IRC-50 (H⁺ form) adsorption, 0.5 N NH₄OH elution, silica gel column chromatography, and preparative thin-layer chromatography, yielding a white powder. Structural elucidation using mass spectrometry (molecular formula: C₁₀H₁₄N₆O₃, confirmed by elemental analysis), spectral data (UV, IR, MS fragmentation, ¹H/¹³C-NMR), and comparison with synthetic 2'-amino-2'-deoxyadenosine identified the compound as 2'-amino-2'-deoxy-β-D-ribofuranosyl adenine—the first report of its natural occurrence. Antimicrobial testing revealed sensitivity in Mycoplasma gallisepticum (Kp-133, S-6), M. pneumoniae, and Acholeplasma laidlawii (A)PG-8 (MIC: 6.25 μg/ml), while bacteria like Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were resistant. This study represents the first report of a nucleoside antibiotic with antimycoplasmal activity from Actinomadura and the natural production of 2'-amino-2'-deoxyadenosine.