Abstract: Objective To establish an improved method for stereotactic location of the supraoptic nucleus in rats. Methods Twenty-four SD rats were randomly divided into experimental group (12 rats) and control group (12 rats) for oblique (20° to the left) stereotactic puncture (OSP group) and vertical stereotactic puncture (VSP group), respectively, both targeting the supraoptic nucleus (SON). The surgical data and postoperative (within 24) mortality of the rats were compared between the two groups. Results The nucleus locating time was longer in OSP group than in VSP group (59.55±3.64s vs 27.44±2.18 s, P= 0.000), and the postoperative mortality rate of the rats did not differ significantly between the groups (0 vs 44.4%, P=0.082). In OSP group, compared with VSP group, the procedure was associated with a lowered rupture rate of the superior sagittal sinus (11.1% vs 88.9%, P=0.003), a shortened hemostatic time after craniotomy (52.89±24.05 s vs 157.445 ime a s, P=0.000) and after puncture (24.33 reas 45 s vs 133.89±28.81 s, P=0.000), and also a shortened operation time (178.89 on tims vs 362.44 timees, P= 0.000). Conclusion The improved method for locating supraoptic nucleus in rats is convenient, stable and reproducible, and helps to avoid important blood vessels and specific nuclei according to the needs of different experiments and allows the operators to choose different surgical paths.