The gut microbiota (GM) and its metabolites are central regulators of mucosal immunity in both the gut and the airways - a concept dubbed 'gut-lung axis'. Changes in GM composition facilitates respiratory diseases and infections. The 'gut-lung axis' concept in birds remains undefined despite increasing societal demands for sustainable health solutions in livestock. Since the majority of pathogens causing infectious diseases in poultry target the gut and/or the airways, understanding the contribution of the 'gut-lung axis' to the chicken immune system is crucial for implementing measures to improve animal robustness in commercial poultry. Here we collected caeca, caecal contents, spleen and lung samples from conventional (CV) and germ-free (GF) chickens to assess how the GM regulates transcriptomic (RNA-seq) and metabolic (H1-NMR) signatures along the 'gut-lung axis'. As expected, the caeca and its contents in GF birds show a dramatically altered physiological landscape, suggestive of deficiencies in cell cycle in parallel to the lack of key GM-derived regulatory metabolites, such as short-chain fatty acids (SCFA). We also reveal the functional existence of the 'gut-lung axis' in chickens, with the detection of SCFA in peripheral compartments of CV birds and the significant differential expression of genes related to innate immunity in the caeca, spleen and lungs. This latter observation was expanded in vitro, where the SCFA butyrate possess antiviral functions in chicken respiratory epithelial cells via IFN-I-related pathways. Further research will help define immunoregulatory checkpoints to be exploited in poultry via GM modulation strategies, such as tailored feed additives and probiotics.