The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17% of the human genome and retain mobility properties within the genome. At present, 80-100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.