Systemic lupus erythematosus (SLE) is an autoimmune disease associated with increased T cell activity. Dual-specificity phosphatase 22 (DUSP22), a member of the protein tyrosine phosphatase family, regulates T cell activation by deactivating a specific site on lymphocyte-specific protein tyrosine kinase (Lck). Research on DUSP22 knockout mice has revealed that lacking DUSP22 enhances T cell-mediated immune responses, suggesting a potential association with SLE. Recent findings have identified specific gene variants of DUSP22 in DNA samples from SLE patients, corresponding to the protein mutations S36Y, H38Y, D39Y, and R80H. In this study, we used circular dichroism (CD) spectroscopy, para-nitrophenyl phosphate (pNPP) activity assays, nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography to investigate the effects of these SLE-associated DUSP22 mutants. CD analysis results indicated no significant differences in secondary structure. However, notable differences were observed in phosphatase activity. The activity (based on Kcat) of the SLE-related mutants decreased in the order of tDUSP22-WT ≈ tDUSP22-R80H > tDUSP22-H38Y > tDUSP22-D39Y >> tDUSP22-S36Y. Chemical shift perturbation (CSP) experiments revealed that tDUSP22-D39Y showed the most changes around the active site, notably with the loss of resonance in the D-loop. Additionally, high CSPs were observed for A56 and D57 in tDUSP22-H38Y. The SLE-associated mutants tDUSP22-S36Y, H38Y, D39Y, and R80H demonstrated a structure-activity relationship: residues closer to the active site or with more interactions show greater reductions in activity and more pronounced conformational changes upon mutation. Thus, tDUSP22-S36Y and tDUSP22-D39Y are considered more severe variants in SLE patients. Furthermore, the significance of the affinity changes of SLE mutants for pNPP remains unclear, warranting further investigation into the interactions between these SLE-related mutants and their physiological substrate, Lck, to better understand their role in SLE progression.