This study investigated the cytotoxicity of gemcitabine using the marine ciliate Euplotes vannus as the test organism. The median lethal concentrations ( LC 50 values) were determined using acute toxicity tests within an exposure time of 30 min with 0, 6, 12, 24, and 48 mg mL −1 gemcitabine. The median inhibition effect ( IC 50 value) on the growth of the ciliate cells was examined using chronic toxicity tests within 5 days (120 h) after exposure for 30 min with 0, 0.7, 3.5, 7, and 14 mg mL −1 gemcitabine. The 30-min LC 50 value was 10.66 mg mL −1. The LC 50 values decreased with increasing exposure times and well fitted to the toxicity curve equation LC 50 = 10.93 + 28.4e −0.19 t ( R 2 = 0.93; P < 0.05, t = exposure time). The IC 50 value for growth rates was 7.05 mg mL −1, and the inhibition effect on growth rates well fitted to the model equation r % = 0.8681e −0.0782 Cgem ( r % means growth rate with inhibition by gemcitabine, C gem means concentrations of gemcitabine, R 2 = 0.99 and P < 0.05). The LC 50 values of a wide range of gemcitabine concentrations could therefore be predicted for any given exposure time. These results suggest that the clinical dose of gemcitabine (20 mg mL −1) was higher than the 30-min LC 50 value, which was almost the same as the 6-min LC 50 value (19.88 mg mL −1) for E. vannus cells. The results also demonstrate that E. vannus can be used as a robust test organism for bioassays of chemotherapeutic drugs during short exposure periods.