Colonization of Carya cordiformis sapwood by Ceratocystis smalleyi and subsequent host defence responses following artificial inoculation were investigated using anatomical and histological techniques. Hyphae of C. smalleyi were observed in all sapwood xylem features confirming the ability of the pathogen to invade and colonize the xylem tissues of the host species. The fungus was isolated from within and at the margins of discoloured sapwood areas at 2 and 12 months after inoculation. General host defence responses that included vessel occlusion with gels or tyloses, lipid accumulation, and production of phenolic compounds were observed in xylem tissues of inoculated C. cordiformis stems. Pectic substances, lipids, and to a rare extent, phenolic compounds were detected in vascular gels. The lipid-rich barriers observed likely prevent lateral expansion of the fungus in the sapwood. Furthermore, lack of fungus sporulation within vessels may restrict axial spread of the fungus. C. smalleyi appears to be a limited vascular wilt pathogen of bitternut hickory based on these observations and previously reported sap flow reduction correlated with multiple infections in artificially inoculated trees.