Eudistomin C (EudiC), a natural product, shows potent antitumor and antiviral activities, but the target molecule and the mechanism of action remain to be revealed. Here, we show that the 40S ribosome is the target in EudiC cytotoxicity. We isolated EudiC- resistant mutants from a multidrug- sensitive yeast strain, and a genetic analysis classified these YER (yeast EudiC resistance) mutants into three complementation groups. A genome- wide study revealed that the YER1- 6 mutation is in the uS11 gene (RPS14A). Biotinylated EudiC pulled down Rps14p- containing complexes from 40S and 80S ribosomes, but not from the 60S ribosome. EudiC strongly inhibited translation of the wild- type strain but not of YER1- 6 in cells and in vitro. These results indicate that EudiC is a protein synthesis inhibitor targeting the uS11- containing ribosomal subunit, and shows cytotoxicity by inhibiting protein translation.