A culture identified as Streptomyces karnatakensis was found to produce a novel cyclic hexadepsipeptide antibiotic designated A83586C. The structure was elucidated by X-ray crystallography, and full 1H and 13C NMR assignments are reported. The absolute configuration was confirmed by the detection of D-threonine in the acid hydrolysate of A83586C. A83586C had potent Gram-positive activity in vitro but lacked in vivo efficacy in mice.