3′-Sialyllactose (3′-SL), a natural prebiotic, maintains immune homeostasis and exerts anti-inflammatory and anti-arthritic effects. Although regulatory T cells (Tregs) prevent excessive inflammation and maintain immune tolerance, the effect of 3′-SL on Treg regulation is unclear. This study aimed to investigate the effect of 3′-SL on Treg responses in atopic dermatitis (AD) pathogenesis. Oral administration of 3′-SL reduced AD-like symptoms such as ear, epidermal, and dermal thickness in repeated topical application of house dust mites (HDM) and 2,4-dinitrochlorobenzene (DNCB). 3′-SL inhibited IgE, IL-1β, IL-6, and TNF-α secretion and markedly downregulated AD-related cytokines including IL-4, IL-5, IL-6, IL-13, IL-17, IFN-γ, TNF-α, and Tslp through regulation of NF-κB in ear tissue. Additionally, in vitro assessment of Treg differentiation revealed that 3′-SL directly induced TGF-β-mediated Treg differentiation. Furthermore, 3′-SL administration also ameliorated sensitization and elicitation of AD pathogenesis by suppressing mast cell infiltration and production of IgE and pro-inflammatory cytokines in mouse serum by mediating the Treg response. Furthermore, Bifidobacterium population was also increased by 3′-SL administration as prebiotics. Our data collectively show that 3′-SL has therapeutic effects against AD progression by inducing Treg differentiation, downregulating AD-related cytokines, and increasing the Bifidobacterium population.