Asthma is an inflammatory airway disease characterized by increased infiltration of inflammatory cells into the airways and poor respiratory function. Bufalin is one of the biological ingredients obtained from Chansu. Bufalin was found to possess various pharmacological properties including anti-inflammatory activities. However, the effect of bufalin treatment on asthma has not yet been reported. Therefore, this study aimed to investigate the inhibitory effect of bufalin on asthmatic response in a murine model. A mouse asthma model was developed by ovalbumin (OVA) sensitization and challenge in the BALB/c mice. OVA-specific serum IgE and the levels of interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluid (BALF) were determined by an enzyme-linked immunosorbent assay. Recruitment of inflammatory cells into BALF or lung tissues, and goblet cell hyperplasia were evaluated by histological staining. The expression levels of inhibitory subunit of nuclear factor-kappa B (NF-κB) alpha (IκBα) and phosphorylated p65 protein were measured by Western blot analyses. The results demonstrated that bufalin (5 and 10 mg/kg) markedly attenuated hyperresponsiveness, and strongly suppressed the OVA-induced increases of total inflammatory cells including macrophages, eosinophils, lymphocytes, and neutrophils in BALF. The levels of IL-4, IL-5, and IL-13 in BALF and OVA-specific IgE in serum were significantly reduced by bufalin. Histological staining of lung tissues showed that bufalin reduced inflammatory cell infiltration and goblet cell hyperplasia. The results of Western blotting indicated that bufalin suppressed the IκBα degradation from NF-κB, and reduced the level of phosphorylated p65 protein in the lung tissues. These data suggest that bufalin can exert its anti-inflammatory effects possibly through the inhibition of the NF-κB activity.