Objective: 2-D08, a SUMO E2 inhibitor, has several biological functions, including anti-cancer effects. However, the effects of 2-D08 in uterine leiomyosarcoma (Ut-LMS) are unknown. This study explored the anti-cancer activity of 2-D08 against human Ut-LMS cells. Methods: We used SK-LMS-1 (human Ut-LMS cells) as an in vitro model. Cell viability (MTT assay), colony formation, Ki67 staining, and BrdU proliferation assays were used to assess the effect of 2-D08 on cell proliferation. Caspase-3 activity was assessed by measuring 2-D08-induced apoptosis. Western blot analysis and immunoprecipitation were used to assess the influence of 2-D08 on cellular signaling. Results: 2-D08 inhibited cell viability in a dose- and time-dependent manner in the SK-LMS-1 cells. Treatment with 2-D08 significantly inhibited the colony-forming ability of SK-LMS-1 cells. Using Ki67 staining and BrdU assay, we found that 2-D08 treatment had anti-proliferative effects on SK-LMS-1 cells. The proliferation of SK-LMS-1 cells was also suppressed by 2-D08 through upregulation of p21, though 2-D08 did not further promote Caspase-3 activation and apoptosis. In addition, 2-D08 did not directly induce smooth muscle phenotypic switching in SK-LMS-1 cells, as assessed by the expression of α-SM-actin, TAGLN, and calponin 1. Additionally, proteasome inhibition increased the steady-state levels of p21 in SK-LMS-1 cells, and 2-D08 regulates these signaling pathways. Conclusion: These results indicate that in the SK-LMS-1 cells, 2-D08 inhibits cell proliferation by regulating the stabilization of p21. Therefore, 2-D08 may be a promising candidate for human Ut-LMS. However, the appropriate dose of this compound should be carefully selected