Objective：To develop an in vitro model for bovine dentinal tubule infection of root canals and to investigate dentinal tubules invasion by Enterococcus faecalis. Methods：Thirty-six freshly extracted，mature bovine incisors were used in this study（six of them for the control at random，others for the test）. Following biomechanical instrumentation and autoclaving，all the specimens were inoculated with Enterococcus faecalis suspension. To investigate the bacterial status in infected tubules，the images stack were ob－tained by scanning electron microscopy（SEM） in different time intervals，which were one，two，three，four，and eight weeks for the test and eight weeks for the control. Results：With the development of time，infected dentinal tubules increased. The initial time of denti－nal tubules invaded by Enterococcus faecalis was two weeks，and the dense invasion time was four weeks，but there was no significant difference between four weeks and eight weeks. They were in interval distribution，with densely packed in some tubules whilst empty in the adjacent tubules. The bacteria also invaded orthodentine at the same time，and they were blocked. Conclusion：Enterococcus faecalis can penetrate into dentinal tubules and orthodentine，and it takes four weeks to establish this in vitro model under anaerobic condition.