BACKGROUND: Plasmacytoid dendritic cells (pDCs) are previously reported to induce immune tolerance to allergen by inhibiting allergic Th2 cell priming. However, there is limited knowledge on pDC function during Th2 effector phase of allergic asthma. OBJECTIVE: We aimed to investigate the role of pDCs in ongoing allergic asthma following airway sensitization and challenge with house mite dust (HDM). METHODS: BDCA2-DTR mice were used for pDC depletion. RNA-sequencing (RNA-seq) of sorted mouse lung pDCs, in vitro cell experiments, and in vivo CCR2 blockade or Tβ4 supplementation were performed to elucidate the underlying mechanism. RESULTS: pDC depletion during HDM challenge enhanced CCR2-dependent inflammatory monocyte-derived cells (inf-MCs) accumulation, leading to exacerbated Th2-mediated allergic asthma phenotypes. RNA-seq analysis revealed an anti-inflammatory peptide thymosin β4 (Tβ4) among the most up-regulated genes in asthmatic lung pDCs. Airway epithelial cells-derived IL-33 was required for up-regulating Tβ4 expression in pDCs, which represented the main source of Tβ4 in asthmatic lungs. Importantly, Tβ4 supplementation reversed the exacerbation of asthmatic phenotypes in BDCA2-DTR mice. Alveolar macrophages were identified as the major source of CCL2 and the target of Tβ4 in asthmatic lungs. Mechanistically, Tβ4 inhibited IL-4/IL-13-induced phosphorylation of JAK1 and STAT6 and downstream EGR2 expression in macrophages, thereby reducing CCL2 expression and subsequent inf-MC recruitment. Unexpectedly, decreased serum Tβ4 levels were detected in mice and humans with ongoing allergic asthma. This could be due to increased uptake of Tβ4 by alveolar macrophage, which was required for its inhibitory effect on CCL2 expression. CONCLUSIONS: Lung pDCs exert anti-inflammatory effects in allergic asthma via expressing Tβ4, which could have therapeutic potential.