The stress-strain relationship is determined by the inherent mechanical properties of the intestinal wall, the geometric configurations, the loading conditions and the zero-stress state of the segment. The purpose of this project was to provide morphometric and biomechanical data for rat duodenum, jejunum and ileum. The circumferential strains were referenced to the zero-stress state. Large morphometric variations were found along the small intestine with an increase in the outer circumferential length and luminal area and a decrease in wall thickness in distal direction. The serosal residual strain was tensile and decreased in distal direction (P<0.05). The mucosal residual strain was compressive and the absolute value decreased in distal direction (P<0.001). The stress-strain experiments showed that the duodenum was stiffest. All segments were stiffest in longitudinal direction (P<0.05). In conclusion, axial variation in morphometric and biomechanical properties was found in the small intestine. The Zero-stress state must be considered in future biomechanical studies in the gastrointestinal tract.