Real-world evidence on clinical and molecular outcomes of systemic therapy for pediatric atopic dermatitis remains limited. Within the prospective TREATkids registry, we conducted an observational analysis of children and adolescents treated with dupilumab in routine care. Baseline data from 200 and follow-up data from 124 patients were evaluated for clinician- and patient-/caregiver-reported outcomes, alongside epidermal proteomic profiling using tape strips and the Olink Explore Inflammation 384 (n = 20) panel and 16S ribosomal RNA gene sequencing for skin microbiome assessment in subsets (n = 48). At treatment initiation, disease burden was high (mean Eczema Area and Severity Index = 16.5, objective SCORing Atopic Dermatitis = 44.9, peak pruritus numerical rating scale = 6.6). By month 3, Eczema Area and Severity Index 50, 75, and 90 response rates were 87, 60, and 30%. Response rates at months 6 and 12 were generally consistent with those observed at month 3, with no discontinuations and conjunctivitis occurring in 4.0%. Proteomic analyses demonstrated marked baseline upregulation of alarmins, T helper 2 chemokines, and tissue-remodeling markers in lesional skin, followed by downregulation of 144/161 dysregulated proteins at month 3, including CCL17/TARC, CXCL8, IL-6, IL-18, and matrix metalloproteinases. Microbiome profiling showed baseline dysbiosis with Staphylococcus aureus overabundance and reduced α-diversity, normalizing toward a nonlesional-like state after therapy at month 3. Overall, dupilumab was associated with rapid, sustained clinical and molecular improvement.