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Amid: The aim of this study was to develop a convenient method for isolating Lactobacillus without anaerobic equipments.Method and Results: Glass bottles (250 ml in size) were used to contain the commercial MRS media supplemented with agar power.After the media (about 150-ml each bottle) was dropped to the bottle,carbon dioxide (CO2) gas was aerated into the bottle for about 10 minutes to exhaust oxygen,and then the bottle was autoclaved at 115℃for 10 min.After sterilization,the mouth of bottle was sealed with sealing film and the bottle was placed obliquely at room temperature until the media was solid.A bottle with solid slant medium was made for isolation of Lactobacillus.Four different species of Lactobacillus were isolated from pig ilea digesta using this new method.Conclusion: This report provided a new simple method to isolate Lactobacillus,which didnt require anaerobic equipments needed in other methods.Significance and Impact of the Study: This new method provides a simple and useful tool to isolate Lactobacillus and has the potential to be used as a tool to isolate other anaerobes.