The STAT3 is an important regulator in a wide range of different cell types. Human variants are associated with several immune dysregulation diseases. The current study investigated the clinical, genetic, and immunobiological data obtained from a family with novel heterozygous variants located at p.Y360C of the DNA binding domain. The clinical manifestations of these patients include autoimmunity, immunodeficiency, and postnatal growth defects. Broad STAT3 regulated cells including patient primary immune cells and HEK293 cells harboring the variant were assessed. Remarkably high levels of STAT3-regulated cytokines were detected in the sera of the patients. STAT3 nuclear binding and STAT3 activity were higher in STAT3-transduced HEK293 cells containing the p.Y360C variant when compared to HEK cells expressing wild type (WT) STAT3. Upon cytokine activation, variants inhibited nuclear translocation of the WT STAT3 molecule. We also demonstrated that PBMCs from these patients exhibit significantly higher mitochondrial activity compared to that of healthy controls. The exploration of the effects of Y360C variants described in our study provides novel insights into the molecular effects of the STAT3 variant and its role in the pathophysiology of gain-of-function syndromes.