Zygosporin D (3) and two new cytochalasins (4 and 5) were isolated from the culture filtrate of the fungus Metarrhizium anisopliae and characterized on the basis of their spectral data and chemical conversions. The new cytochalasins, 4 and 5, were determined to be deacetylcytochalasin C and (6R,16S,18R,21R)-18,21-dihydroxy-16, 18-dimethyl-10-phenyl[11]cytochalasa-13(E),19(E)-diene-1,7,17-trio ne, respectively. Of these three cytochalasins, only zygosporin D is an effective inhibitor of shoot elongation of rice seedlings.