Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infection due to host restriction. Current research about host restriction mainly focuses on the barrier of cell membrane, nuclear envelope, and host proteins, whether microRNAs (miRNAs) play a role of host restriction is largely unknown. Herein, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in the host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two sus scrofa (ssc-, swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222 Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial cells (NPTr) and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus.IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication, however, the role of miRNAs in host species specificity is unclear. Here we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells are modulated by NF-κB P65 phosphorylation in response to AIV infection, but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.