Background: Sepsis is still developing exorbitantly high mortality. T cell activation plays a crucial role in sepsis’s initial innate immune reactions. The imbalance between Th17 and Treg participates in sepsis progression. CD28 signaling pathway was essential for the expression of inflammatory cytokines related to Th17, and play a crucial role in the maintenance of Treg. Methods: 60 sepsis patients’ baseline conditions were recorded, and the expressions of inflammatory factors in the peripheral blood and levels of procalcitonin (PCT) were detected. PBMCs were separated, subtypes of T cells and related biomarkers were measured by FACS. Furthermore, the relationship between the above indicators and patients’ condition scoring (APACHEⅡand SOFA) and ICU hospitalization time were analyzed. To investigate effects of CD28 on the balance of Th17 between Treg, anti-CD28 antibody was intraperitoneal administrated to cecal ligation and puncture (CLP) mice. Results: Compared with septic patients who stay in ICU more than 14 days, the Th17/Treg ratio of patients fewer than 14 days was significantly lower. Moreover, the expression of CD28 was significantly higher in sepsis patients than that of healthy donors. After administration of CD28 monoclonal antibody, 7-day mortality and clinical score were significantly improved in septic mice, with splenocyte Th17/Treg ratio decreased. CD28 antibody alleviates the expression of pro-inflammatory factors and spleen injury related to apoptosis. Conclusions: Th17/Treg ratio revealed septic patient severity and as a predictor of ICU stay. CD28 monoclonal antibody could improve 7-day mortality of septic mice by decreasing T cell apoptosis and amending the ratio of Th17/Treg.