Iejimalides (IEJLs) A–D are 24-membered macrolides isolated from a tunicate Eudistoma cf. rigida , and exhibit potent cytotoxicity in vitro and antitumor activity in vivo . We previously reported that the molecular target of IEJL-A and -B was the vacuolar-type H⁺-ATPases (V-ATPases). However IEJL-C and -D, which are sulfonylated IEJL-A and -B, respectively, show more potent antitumor activity, and their molecular targets remain to be discovered. Here, we report that IEJL-C is also a potent V-ATPase inhibitor by binding in a site similar to the bafilomycin-binding site. Two-hour treatment with IEJL-C resulted in the complete disappearance of acidic organelles in HeLa cells. Interestingly, after 24-h treatment, small actin aggregates were observed instead of actin fibers. The same actin reorganization was also observed in cells treated with another V-ATPase inhibitor, bafilomycin A₁. Because IEJLs did not inhibit actin polymerization in vitro , these results suggest that the primary target of IEJL-C, as well as IEJL-A and -B, is V-ATPase, and actin reorganizations are probably caused by the disruption of pH homeostasis via V-ATPase inhibition.