A new nanaomycin analog, nanaomycin K, was isolated from a cultured broth of actinomycete strain "Streptomyces rosa subsp. notoensis" OS-3966. Nuclear magnetic resonance (NMR) analyses revealed that the planar structure of nanaomycin K had an ergothioneine moiety. To determine the absolute configuration, nanaomycin K was semi-synthesized using standards of nanaomycin E and L-ergothioneine. The natural and semisynthetic nanaomycin K were identified as the same compounds based on retention time, mass spectrometry, H-1 NMR, and optical rotation data. Nanaomycin K showed cytotoxicity against Madin-Darby canine kidney (MDCK) cells undergoing transforming growth factor (TGF) beta 1-induced epithelialemesenchymal transition. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.