This study aimed to investigate the effects of L-Asparaginase (L-ASP) on leukemia cell proliferation, cell cycle distribution and geminin expression. Jurkat cells were treated with different concentration of LASP for 24 h, 48 h or 72 h. Cell proliferation was analysed by the CCK-8 assay, cell cycle distribution was examined by flow cytometry, and mRNA and protein expression levels of geminin were assessed by PCR and Western blot analysis. The results showed that L-ASP inhibited the proliferation of Jurkat cells in a dose- and time-dependent manner. In addition, L-ASP at 0.5-10 U/ml induced G1 phase arrest of Jurkat cells. Geminin mRNA and protein expression remarkably decreased in Jurkat cells treated with L-ASP at 1.0-10 U/ml. In conclusion, our study suggests that the anti-leukemia mechanism of LASP is related to the arrest of leukemia cells in G1 phase and the downregulation of geminin expression.