We aimed to investigate the effect of miR-506-3p on the proliferation and apoptosis of airway smooth muscle cells (ASMCS) in asthmatic mice by regulating the activation of TLR4/NF-κB signaling pathway through targeted regulation of C-C Motif Chemokine Ligand 2 (CCL2) expression. Twenty-four BALB/c mice of specific pathogen-free grade were selected to establish asthmatic mouse model, which were randomly divided into normal control group and asthma model group (n = 12 for each group). HE and IHC staining, bioinformatics and dual luciferase reporter assay, RT-PCR MTT, flow cytometry and Western blot were used in this research. HE staining showed airway epithelium thickening, submucosal inflammatory cell infiltration and airway smooth muscle thickening, and the positive expression rate of CCL2 was significantly increased in asthma model group (all P < 0.05). CCL2 was the target gene of miR-506-3p. Moreover, the expression of miR-506-3p in asthma model group was significantly decreased, the mRNA and protein expression levels of CCL2, TLR4, NF-κB (p65) and Bcl-2 were significantly increased, while those of Bax were decreased (all P < 0.05). In miR-506-3p mimic group or siRNA-CCL2 group, the expression of CCL2, TLR4, NF-κB (p65) and Bcl-2 decreased obviously, while that of Bax increased, cell proliferation decreased, G1 phase prolonged, G2 & S phases shortened, and apoptosis rate increased significantly (all P < 0.05), whereas the opposite trends were found in miR-506-3p inhibitor group (all P  0.05). Overexpression of miR-506-3p can inhibit ASMCS proliferation and promote apoptosis via inhibiting CCL2 expression and suppressing the activation of TLR4/NF-κB signaling pathway. Inhibited expression of miR-506-3p can reverse the positive role of CCL2 gene silencing. Our study is the first to prove the beneficial role of miR-506-3p-CCL2-TLR4/NF-κB regulatory axis in the development of asthma.