Carbon dioxide (CO2) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO2 (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. The current state of the art is that hypercapnia is detrimental in the context of a pathogenic infectious challenge (1), however, it may be beneficial against a background of destructive inflammation (2). Thus, CO2 can act at the axis of immunity and inflammation through its role as a signaling molecule contributing to suppression of innate immune responses and attenuation of pro-inflammatory signaling. The molecular mechanisms underpinning CO2-dependent modulation of immune/inflammatory signaling are not fully understood but are at least in part regulated through modulation of the NFκB pathway. NFκB is a master regulator of immune and inflammatory signaling (3). We have recently demonstrated a CO2-dependent cellular re-organisation of NFκB family members, particularly those associated with non-canonical NFκB signaling (IKKα (4) and RelB (5)). In this study, we characterize the CO2-dependent modulation of RelB, identifying a CO2-dependent cleavage from its C-terminal that is dependent on a 20 amino-acid motif. Furthermore, using semi-quantitative mass spectrometry, we characterize the RelB interactome under conditions of elevated CO2 to examine CO2- dependent changes in protein-protein interactions and gain mechanistic insight into the molecular mechanisms underpinning CO2 sensitivity of the NFκB pathway. A better understanding of the molecular mechanisms underpinning CO2 signaling in the context of inflammation and immunity could be of clinical benefit regarding the treatment of patients experiencing hypercapnia and for the development of new therapeutic strategies for the manipulation of inflammatory signaling.