Objective: Foxp3⁺ regulatory T cells (T_reg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of T_reg cells. To further define the mechanism of CaMK4 on T_reg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4. Methods: We generated MRL/lpr Foxp3-GFP mice to record T_reg cells; stimulated naïve CD4⁺ T cells from MRL/lpr Foxp3-GFP mice under T_reg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of T_reg cells in the kidney. Results: We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes T_reg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of T_reg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased T_reg cells in target organ. Conclusion: Our results indicate that KN-93 treatment enhances the generation of T_reg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.