Metolachlor is one of the most commonly used herbicides in the United States. Protein synthesis is inhibited when roots and shoots of susceptible plants absorb this synthetic herbicide. While quite effective in killing weeds, several studies have shown that exposure to metolachlor results in decreased cell proliferation, growth and reproductive ability of non-target organisms. However, the mode of metolachlor action in non-target organisms has not yet been elucidated. The current study assessed effects of metolachlor exposure on immortalized human liver (HepG2) cells. Results from cell proliferation assays showed that a 72-h exposure to 50 parts per billion (ppb) metolachlor significantly inhibited growth of these cells compared to untreated controls while a decrease in the cell division rate required exposure to 500 ppb metolachlor for 48 h. Flow cytometry analysis of cell cycle distribution revealed that 500 ppb metolachlor treatment resulted in fewer HepG2 cells in G2/M phase after 72 h. Real-time PCR analysis showed a significant decrease in the abundance of the cyclin A transcripts after 12 h in cells exposed to 300 ppb metolachlor. These results suggest metolachlor may affect progression through the S phase of the cell cycle and entrance into the G2 phase.