Objective To investigate TFF3 (rTFF3) protein expression profile in the adult and embryonic Wistar rats by using indigenous anti-rTFF3 polyclonal antibody and to explore the effect of TFF3 on the early embryonic development and its molecular pattern(s) existing in the intestinal mucosa. Method Anti-rTFF3 polyclonal serum was raised from the immunized New Zealand rabbit by synthetic N-terminal peptide of rTFF3. rTFF3 protein in the tissues was then detected by the self-made anti-rTFF3 polyclonal antibody with the immunohistochemical method. rTFF3 protein in the intestinal mucosa was detected by SDS/PAGE and Western blot. Result The anti-rTFF3 polyclonal antibody was sensitive and specific to rTFF3 molecule. rTFF3 was expressed extensively in the mucosa of small intestine, large intestine and epithelium of bile duct of adult Wistar rats, and it was also present in the late embryonic intestinal mucosa (two-week gestation and without mature goblet cell in the intestine). rTFF3 existed in the form of complexes, molecular weights of which were 250kD and 55kD respectively. Conclusion rTFF3 protein is located mainly in the goblet cells of the intestine. Its presentation seems to precede the maturation of goblet cells, and it might be involved in embryonic development. The majority of active rTFF3 molecules exist in a complex form, which might interact with unknown protein(s), and only a minor portion is in the form of a monomer.