Objective To study the stress-strain properties of intestine in vivo. Methods The segments from distal ileum were studied in 16 female Wister rats with 10 rats in in vivo group and 6 rats in in vitro group. Papaverine was injected into the peritoneal cavity to relax the intestine. The pressures were applied from zero to 8 cmH 2O with 1 cmH 2O stepwise. After finishing the distension test, tissue rings and strips were cut from the segments for acquiring the no-load and zero-stress state. The stresses and strains were computed using the definitions by Kirchhoff and Green according to the morphometric data obtained from the photographic images. Results (1) The circumferential and longitudinal stress-strain curves both in vivo and in vitro fitted well to mono-exponential function: τ=(τ *+β)e α(ε-ε *) -β (determination coefficients>0.96). (2) The stress-strain curves in circumferential direction shifted to the right compared to the curves in longitudinal direction. The curves in vivo were steeper than the curves in vitro both in circumferential and in longitudinal directions. (3) Comparing the stress-strain curves with the zero-stress state as reference, the curves referenced to the no-load or zero-pressure state shifted to the left in circumferential direction and to the right in longitudinal direction. The differences between the curves were significant (P<0.05). Conclusion (1) The ileum in vivo showed a high degree of non-linearity and anisotropy. The intestine was stiffer in vivo than in vitro, and stiffer in longitudinal direction than in circumferential direction. (2) It is important to use zero-stress state as the initial reference for the stress-strain analysis.