Pellino-1 has recently been identified as a regulator of interleukin-1 (IL-1) signaling, but its roles in regulation of responses of human cells to human pathogens are unknown. We investigated the potential roles of Pellino-1 in the airways. We show for the first time that Pellino-1 regulates responses to a human pathogen, rhinovirus minor group serotype 1B (RV-1B). Knockdown of Pellino-1 by small interfering RNA (siRNA) was associated with impaired production of innate immune cytokines such as CXCL8 from human primary bronchial epithelial cells in response to RV-1B, without impairment in production of antiviral interferons (IFN), and without loss of control of viral replication. Pellino-1 actions were likely to be independent of interleukin-1 receptor-associated kinase-1 (IRAK-1) regulation, since Pellino-1 knockdown in primary epithelial cells did not alter responses to IL-1 but did inhibit responses to poly(I.C), a Toll-like receptor 3 (TLR3) activator that does not signal via IRAK-1 to engender a response. These data indicate that Pellino-1 represents a novel target that regulates responses of human airways to human viral pathogens, independently of IRAK signaling. Neutralization of Pellino-1 may therefore provide opportunities to inhibit potentially harmful neutrophilic inflammation of the airways induced by respiratory viruses, without loss of control of the underlying viral infection.