Atopic dermatitis (AD) is a persistent and recurring inflammatory condition affecting the skin. An expanding corpus of evidence indicates the potential participation of transforming growth factor-β1 (TGF-β1) in the modulation of inflammation and tissue remodeling in AD. The primary objective of this study was to examine the aberrant modulation of TGF-β1/small mothers against decapentaplegic homolog 3 (SMAD3) signaling through a comprehensive analysis of their molecular and protein expression profiles. The study encompassed an aggregate of 37 participants, which included 25 AD patients and 12 controls. The assessment of mRNA and protein levels of TGF-β1 and SMAD3 was conducted utilizing quantitative real-time PCR and immunohistochemistry (IHC), whereas serum IgE and vitamin D levels were estimated by ELISA and chemiluminescence, respectively. Quantitative analysis demonstrated a 2.5-fold upregulation of TGF-β1 mRNA expression in the lesional AD skin (P†<†0.0001). IHC also exhibited a comparable augmented pattern, characterized by moderate to strong staining intensities. In addition, TGF-β1 mRNA showed an association with vitamin D deficiency in serum (P†<†0.02), and its protein expression was linked with the disease severity (P†<†0.01) Furthermore, a significant decrease in the expression of the SMAD3 gene was observed in the affected skin (P†=†0.0004). This finding was further confirmed by evaluating the protein expression and phosphorylation of SMAD3, both of which exhibited a decrease. These findings suggest that there is a dysregulation in the TGF-β1/SMAD3 signaling pathway in AD. Furthermore, the observed augmentation in mRNA and protein expression of TGF-β1, along with its correlation with the disease severity, holds considerable clinical significance and emphasizes its potential role in AD pathogenesis.