Various oncogenes correlate with tumor phenotypes, which include transformed morphology, loss of contact inhibition, colony formation in soft agar, and tumorigenicity in nude mice. Thus, inducers of phenotypic reversion in oncogene-transformed cells may serve as selective antitumor agents without cytotoxicity. Our screening for such substances led to the isolation of trichostatin D, a new member of the trichostatin family, from the actinomycete Streptomyces violaceusniger. Here, we report the fermentation, isolation, structure elucidation, and biological activities of trichostatin D. Structural analysis revealed that trichostatin D is an α-D-glucopyranoside of trichostatin A, a known histone-deacetylase inhibitor. Trichostatin D induced flat reversion in NIH3T3 murine fibroblasts transformed with human papillomavirus oncogenes (NIH3T3/T-601), as well as in NIH3T3 cells transformed with YL-ras or hst-1 (NIH3T3/ras, NIH3T3/hst-1). It inhibited colony formation of these transformed cells in soft agar and exerted growth inhibition on NIH3T3/T-601 cells with an IC50 of 33 ng/ml. These results demonstrate that trichostatin D can reverse the tumor phenotypes of transformed cells, suggesting it is a promising candidate for selective anticancer agents.