Objective To investigate the localization method of microinjection of the solitary nucleus in rats using different parameters and to improve its success rate and accuracy. Methods Twenty rats were divided into a vertical injection group, a Bregma-based localization group, and a Lambda-based localization group according to the experimental progress. The injection point of the vertical injection group was 0. 5 mm at the anterior edge of the skull base of the rat, with a needle insertion depth of 7. 5 mm. The Bregma positioning group used Bregma-11. 0 mm and 0. 5 - 0. 7 mm beside the bilateral midline as the puncture point. The oblique insertion angle was 24° backwards and the depth was 8. 6 mm at the injection point. The Lambda positioning group used Lambda-3. 2 mm and 0. 5 - 0. 7 mm beside the bilateral midline as the puncture point. The oblique insertion angle was 24° backwards and depth was 9. 6 mm at the injection point. The positions of the fluorescent markers in the coronal section of the whole brain after injection of each group of rats were observed. Results The fluorescent label in the vertical injection group was located in the choroid plexus. The fluorescent labeling was unstable in the Bregma-based localization group, and was located in the coronal sections of the cerebellum, superficial brainstem, and choroid plexus. In the Lambda-based localization group, the fluorescent label position in the rats weighing approximately 300 g was located in the space between the cerebellum and the brainstem, while the rats weighing around 400 g could be accurately labeled in the solitary nucleus. Conclusions In rats of approximately 400 g, oblique puncture at a certain angle based on Lambda as the reference point can enable accurate injection into the nucleus of the solitary tract in rats.