Influenza virus is one of the major respiratory pathogens of humans as well as animals, including equines. There is an increasing evidence that bacterial infections are the most common cause of the death during influenza. In horses also, secondary bacterial pneumonia can lead to death, and surviving horses may take up to six months for the complete recovery resulting in heavy economic loss to the equine industry. Interleukin (IL)-23 mediated innate immune response has been shown to protect the host from various respiratory bacterial infections. However, studies to investigate the role of host and viral factors in the regulation of IL-23 are limited. Endoplasmic reticulum (ER) stress-induced transcription factor CHOP-10 and IFN-β has been shown to participate in the regulation of IL-23. Primary hypothesis for the current study was that influenza A virus (IAV) NS1 protein downregulates the IL-23 expression via inhibition of CHOP-10. In order to test our hypothesis, we infected the RAW264.7 cells - a murine macrophage cell line, and primary murine alveolar macrophage cells either with the wild type Influenza A virus (PR/8/34, PR8) or isogenic mutant virus lacking NS1 (delNS1). Quantitative analysis of mRNA expression revealed a significantly higher mRNA expression of IL23p19, IFN-β and CHOP-10 in delNS1 virus infected cells as compared the PR8 virus infected cells. Additionally, overexpression of CHOP-10 partially restored the expression of IL-23p19 in PR8 virus infected cells and knockdown of CHOP-10 resulted in downregulated expression of IL-23p19 in delNS1 infected cells. Taken together, these results suggest that IAV NS1 protein mediated inhibition of CHOP-10 expression leads to downregulation of IL-23 expression in macrophage cells in-vitro. Similar results were also observed in-vivo using IAV and Streptococcus zoooepidemicus (S. ze) co-infection model. In a co-infection mouse model delNS1 virus co-infection resulted in significantly higher expression of the IL-23 and IL-17. Considering the role of IL-23 in protection against respiratory bacterial pathogens, effect of exogenous supplementation of IL-23 was also investigated in the influenza and S. ze co-infection mouse model. We found that a single intranasal dose of recombinant murine IL-23 significantly improved the survival of mice co-infected with PR8 and S .ze. Overall, our study suggests that IAV infection subverts the IL-23 mediated respiratory innate immune response and restoration of IL-23 could protect from influenza-associated respiratory bacterial infections.