A new staurosporine analog, 10'-5''-[(methoxycarbonyl)amino]-2''-methyl-phenylaminocarbonylstaurosporine (1), together with staurosporine (2), was obtained from the culture broth of Actinomycetes Streptomyces sp. (172614). Their structures were elucidated by comprehensive spectroscopic analysis including UV, MS, NMR, and CD spectra. Pharmacological experiments revealed that 1 and 2 showed significant cytotoxicity against human colon tumor cell HCT-116.