Dupilumab is a monoclonal antibody approved for the treatment of atopic dermatitis (AD); however, its effects on molecular, cellular, and immunological levels remain to be elucidated. In this study, blood and dermal interstitial fluid (ISF) from nonlesional (NL) and lesional (L) skin were collected from eight patients with moderate to severe AD, before (visit 2-v2) and at the end of a 16-week treatment with dupilumab (visit 10-v10). Clinical treatment effect was demonstrated by significantly decreased AD severity scores at the end of treatment. At v10 versus v2, the percentages of CD4+ interleukin-producing cells showed a decreasing trend in ISF L and NL, unbound IL-4 levels in plasma were increased, IL-5 levels in ISF L reduced, and levels of factors involved in anti-inflammatory pathways and re-epithelization increased. At v2, ISF L showed that AD lesions might have altered amino acid pathways and lipid signaling compared to ISF NL. At v10, ISF L exhibited raised levels of long- and very-long-chain fatty acids and lipids compared to v2. Furthermore, dupilumab administration caused reduced expression of miR-155-5p and miR-378a-3p in ISF L. In conclusion, results from the present study provided novel knowledge by linking local immune and metabolic alterations to AD pathogenesis and treatment response.