Recent studies have revealed that YKL-40 is involved in the pathogenesis of asthma. However, its specific mechanism remains unclear. The present study aims to investigate the effect of adenovirus vector-mediated YKL-40 short hairpin RNA (shRNA) on regulation of airway inflammation in a murine asthmatic model. Mice were assessed for airway hyperresponsiveness (AHR), total leukocytes and the percentage of eosinophil cells in bronchoalveolar lavage fluid (BALF). YKL-40 mRNA and protein expression levels were detected using quantitative real-time PCR and western blot assays. Enzyme-linked immunosorbent assay (ELISA) was used to detect YKL-40 and eosinophil-related chemokine expression levels in BALF and serum. Lung histology analyses were performed to evaluate the degree of inflammatory cell infiltration around the airway and airway mucus secretion.YKL-40 shRNA significantly inhibited the YKL-40 gene expression in asthmatic mice. In addition, YKL-40 shRNA alleviated eosinophilic airway inflammation, AHR, airway mucus secretion and decreased the levels of YKL-40 in BALF and serum in a murine asthmatic model. The levels and mRNA expression of IL-5, IL-13 in asthmatic mice lung tissues, eotaxin, and GM-CSF in BALF and serum significantly decreased. Bone marrow signaling molecules including IL-5, eotaxin, and GM-CSF were correlated with decreased levels of YKL-40. The study reveals that YKL-40 could be involved in asthma inflammation by altering bone marrow signaling molecules. YKL-40 gene RNA interference could provide new therapeutic strategies for asthma.