Influenza A and B virus infections both cause a host innate immunity response. Here, we report that the robust production of type I and III interferons (IFNs), IFN-stimulated genes, and pre-inflammatory factors can be induced by influenza B virus infection rather than influenza A virus in alveolar epithelial cells (A549) during early infection. This response is mainly dependent on the RIG-I-mediated signaling pathway. The infection by influenza B virus promotes intense Lys63-linked ubiquitination of RIG-I, resulting in cytokine eruption. It is known that the influenza A virus NS1 protein (NS1-A) interacts with RIG-I and TRIM25 to suppress the activation of RIG-I-mediated signaling. However, the present results indicate that the influenza B virus NS1 protein (NS1-B) is unable to interact with RIG-I but engages in the formation of a RIG-I/TRIM25/NS1-B ternary complex. Furthermore, we demonstrate that the N-terminal RNA-binding domain (RBD) of NS1-B is responsible for interaction with TRIM25, and this interaction blocks the inhibitory effect on RIG-I ubiquitination from the NS1-B C-terminus. Our findings reveal a novel mechanism for the host cytokine response to influenza B virus infection through regulatory interplay between host and viral proteins.