Trichloroethylene (TCE) is a common chemical pollutant that exists in air, soil, and drinking water. TCE exposure is known to cause severe hepatotoxicity; however, the mechanisms underlying TCE hepatotoxicity remain poorly understood. In a previous proteomics study, we found that TCE exposure up-regulated the expression of the inhibitor 2 of protein phosphatase 2A (I2PP2A), a potent and specific endogenous inhibitor of protein phosphatase (PP) 2A, in human hepatic L-02 cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down I2PP2A expression in L-02 cells and explored the potential role of I2PP2A in TCE-induced cytotoxicity. We found that TCE treatment of L-02 cells causes decreased cell viability, increased apoptosis and elevated I2PP2A mRNA and protein levels. TCE-treated L-02 cells were also found to have significantly reduced PP2A activity. Lentivirus-mediated I2PP2A knockdown partially prevented the decrease in viability and increased apoptosis induced by TCE treatment. Knockdown of I2PP2A in TCE-treated L-02 cells also suppressed the inhibition of PP2A activity and prevented caspase-3 activation. These data for the first time demonstrate that the up-regulation of I2PP2A could mediate, at least in part, TCE-induced liver cell toxicity through the inhibition of PP2A activity and caspase-3-mediated pathway, and suggest that I2PP2A may play a crucial role in mediating TCE hepatotoxicity.