To propose a convenient and highly reproducible method for repeat collection of rat cerebrospinal fluid (CSF) samples. Methods: Twenty-five SD rats were randomly divided into control group and experimental group. After anesthesia, the rats in the experimental group were positioned with their heads at an angle of 45° to 90° relative to their bodies. A needle was inserted at the center point between the occipital protuberance and the first cervical vertebra, with the needle tip directed toward the head and inserted slowly parallel to the curve of the rat's head, reaching a depth of approximately 5 mm. CSF was then slowly aspirated, and the sample volume was recorded. Comprehensive assessments were conducted by observing changes in rat body weight and Bederson scores, as well as through Evans blue staining and hematoxylin and eosin (HE) staining, and by measuring interleukin-6 (IL-6) levels in the CSF. Results: In the experimental group, successful first-time sampling was achieved in 14 rats (70.0% ) with a sample volume of (93.30±13.33) μL. Successful consecutive second-time sampling was achieved in 10 rats (50.0%) with a sample volume of (93.00± 19.86) μL. Successful consecutive third-time sampling was achieved in 8 rats (40.0%) with a sample volume of (89.75±7.72) μL. Rats in the group with successful consecutive three-time sampling showed an increase in body weight of (47.6±1.51) g, which was not statistically significant compared to the control group (P>0.05). The Bederson scores of the rats in the experimental group were all 0, indicating no significant neurological deficits. No obvious Evans blue staining or morphological changes in brain tissue were observed. The IL-6 levels in the CSF samples collected during the three samplings were (28.98±4.70) pg/mL, (29.37±6.06) pg/mL, and (30.06± 5.47) pg/mL, respectively, with no statistically significant differences among the three collections (P>0.05). However, an increase in IL-6 levels was observed in CSF samples successfully collected after an initial failed attempt (P<0.05). Conclusion: The method of percutaneous cerebellomedullary cistern puncture for CSF collection requires simple equipment, has a short operating time, can be performed by a single individual, and causes minimal trauma to the animals.