Aim This study explored the effect of heat inactivation on the immunomodulatory capabilities of two potential host-derived probiotics (GP21 and GP12) on the intestinal epithelial cells (IEPC) derived from Atlantic cod. Methods and Results The cells were isolated from the four segments of the gut, namely anterior intestine (AI), mid-intestine (MI), posterior intestine (PI) and rectum (RC). The IEPC cultures were exposed to live or heat-inactivated form of GP21 and GP12 for 24h. The expression profiles of bacterial defence genes and cytokine genes in the probiotics-exposed IEPCs showed differential patterns. Heat inactivation did not drastically affect the immunomodulatory properties of the probiotics, and this was explicitly typified by the stimulated expression of g-type lysozyme, hepcidin, transferrin and metallothionein in both forms of the bacteria. There was no distinct expression pattern of the interleukin genes during bacterial exposure. This was in contrast to the chemokines where the expression of these genes in IEPCs was down-regulated upon exposure to the heat-inactivated probiotics. Although heat inactivation did not drastically affect the immunomodulatory capabilities of the probiotics, the live form elicited higher immune responses in the IEPCs in most cases. Conclusion This study showed that bacterial viability was a contributing influence, but not a major limiting factor on the immune-related functions of the host-derived probiotics in vitro. Significance and Impact of the Study GP21 and GP12 are beneficial host-derived bacteria and could be utilized as candidate probiotics in cod aquaculture.