L-mimosine is a rare plant amino acid extracted from Mimosa or Leucaena?spp., and it has been reported to exhibit antitumor activity in a number of types of cancer. However, the underlying mechanisms remain to be clarified. In the present study, the effect of L?mimosine was investigated in human osteosarcoma cells. A Cell Counting Kit?8 assay and flow cytometry were used for toxicity detection. Hoechst staining and transmission electron microscopy (TEM), in addition to western blot analysis, were used for the examination of the associated mechanisms. The results of the present study indicated that L?mimosine significantly inhibited cell proliferation by inducing cellular apoptosis in osteosarcoma cells. The Hoechst staining results and TEM revealed that nuclear damage increased with the concentration increase in L?mimosine, as did the formation of apoptotic bodies. Additionally, the results of the western blot analysis confirmed that the treatment of cells with L?mimosine was accompanied by increasing expression of cleaved caspase?9. L?mimosine?induced apoptosis was inhibited by the caspase?9 inhibitor Z?LEHD?FMK. In?addition, the extracellular signal?regulated kinase (ERK) signaling pathway was suppressed following treatment with L?mimosine. In conclusion, the results of the present study suggested that L?mimosine induced apoptosis via the mitochondrial apoptotic pathway. The ERK signaling pathway was indicated to be an additional mechanism underlying apoptosis induction. The results provided evidence for the use of L?mimosine as a promising candidate for osteosarcoma therapy.