Lycopene is one of the carotenoids often consumed by humans in their diet. Although lycopene exists mainly in the form of the all E-isomer in foods, the considerable quantity of its Z-isomers is found in the human plasma and liver. This observation suggested that the lycopene all-E-isomer was converted into Z-isomers in the human body. In this study, the Z-isomerization of the all-E-isomer was induced in vitro by the pancreatic juice and small intestinal homogenate of male rats under the conditions of 37 °C, pH = 7.5, nitrogen and darkness, as well as shaking. After 2 hours, the proportion of the all E-isomer decreased to 25% and Z-isomer amounts increased relatively. The converted products were identified as 5, 9, and 11 Z-isomers by electronic absorption spectroscopy and mass spectrometry (MS). The observations from this experiment suggested that the Z-isomerization site of the lycopene all E-isomer was located in the small intestinal wall of the rat.