CKLF1, which exhibits chemotactic activities on a wide spectrum of leukocytes, is up-regulated during the progress of asthma. It plays a vital role in the pathogenesis of pulmonary disease. Here, we report that CKLF1 has the capability to activate the NF-κB signaling pathway leading to the pathological change in the lung. The HEK293-CCR4 cell line, which expressed CCR4 stably, was established and screened. Western blot analysis was performed to determine the expression of NF-κB in HEK293-CCR4 and A549 cells following the C27 (10μg/ml) added in each well at different times. These results showed that C27 (10μg/ml) time-dependently induced the accumulation of NF-κB in the nucleus of HEK293-CCR4 and A549 cells. In addition, CKLF1 plasmid (100μg) injection and electroporation led to the asthmatic change in the lung in mice as shown by HE and PAS staining. Furthermore, it was confirmed that CKLF1 significantly up-regulated the p-IκB expression, decreased the IκB expression, and suppressed the NF-κB expression in the cytoplasm of pulmonary tissue in vivo study. Intriguingly, an enhanced nuclear accumulation of NF-κB was observed in the lung of pCDI-CKLF1 electroporated mice, compared to that in the sham group. Therefore, the NF-κB signaling pathway was involved in the asthmatic change induced by CKLF1, among which CCR4 might play a crucial role.