A new cytotoxic macrocyclic trichothecene calcarisporin B1 (1), and two known compounds roridin H (2) and roridin J (3) were isolated from the cultured mycelia of Calcarisporium arbuscula Preuss. The structure of 1 was determined to be 8alpha-acetoxy roridin H on the basis of spectral data. The cytotoxic activities of 1-3 were evaluated in vitro.