MicroRNAs (miRNAs) are known to play important regulatory roles in host-virus interactions. Avian-origin H3N2 canine influenza virus (CIV) has emerged as the most prevalent subtype among dogs in Asia since 2007. To evaluate the roles of host miRNAs in H3N2 CIV infection, here, miRNA profiles obtained from primary canine bronchiolar epithelial cells (CBECs) and canine alveolar macrophages (CAMCs) were compared between infected and mock-infected cells with the H3N2 CIV JS/10. It was found that the expressions of cfa-miR-125b and cfa-miR-151, which have been reported to be associated with innate immunity and inflammatory response, were significantly decreased in CIV-infected canine primary cells. Bioinformatics prediction indicated that 5´ seed regions of the two miRNAs are partially complementary to the mRNAs of nucleoprotein (NP) and non-structural protein 1 (NS1) of JS/10. As determined by virus titration, quantitative real-time PCR (qRT-PCR) and western blotting, overexpression of the two miRNAs inhibited CIV replication in cell culture, while their inhibition facilitated this replication, suggesting that the two miRNAs could act as negative regulators of CIV replication. Our findings support the notion that some cellular miRNAs can influence the outcome of virus infection, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of H3N2 CIV.