Background: Abnormal proliferation of T cells plays an essential role in the pathogenesis of Systemic lupus erythematosus (SLE). The pharmaceutical effect of Hedyotis Diffusa Willd (HDW) on SLE has been investigated previously. Nevertheless, the biomedical mechanism is still left unclear. Objective: This study has been arranged to evaluate the therapeutic effect of the ethyl acetate fraction of HDW (EAHDW) on lupus mice and explore the potential therapeutic mechanism.Methods: EAHDW was prepared with 80% ethanol reflex extraction followed by successive extraction, and analyzed with HPLC and UPLC-Q/TOP-MS. The potential targets and STAT3 affinity regulators were predicted with network pharmacology. The pharmaceutic effect of EAHDW was studied with MRL/lpr mice. Cytokines and autoantibodies were quantified with ELISA assays. The pathological damage of glomerulus and STAT3 expression in the kidney was detected with histochemical and immunohistochemical techniques. The cell cycle properties in cell proliferation were identified with the flow cytometry. The western blot and dual-Luciferase reporter assay were applied to evaluate translational and transcriptional activity of STAT3,respectively. Results: In this study, the extraction ratio of EAHDW was 2.7±1%, in which 19 ingredients were identified.Network pharmacological analysis showed that the target genes of EAHDW were highly focused on influencing the abnormal T cell proliferation in SLE. EAHDW showed the beneficial effects on pathological changes and STAT3 expression in the glomerulus of lupus mice, and the levels of cytokines and autoantibodies in serum. In cytological study, EAHDW treatment attenuated the transcription and phosphorylation of STAT3, which inhibited T cell proliferation by prolonged S-phase of the cell cycle. A total of 5 compounds in EAHDW exhibited high docking affinity to the DNA-binding site of STAT3. Conclusion: EAHDW could reduce the inflammatory response and inhibit the proliferation of T cells by interfering with the STAT3 signaling pathway, thereby playing a therapeutic effect on SLE.