Abstract: Objective?To explore the effects of retrograde liver perfusion with catheterization via heart on the isolation of primary mouse hepatocytes. Methods?In order to more efficiently isolate primary mouse hepatocytes, we improved the traditional two-step collagenase perfusion method. The liver perfusion catheter was inserted through right atrium and suprahepatic vena cava, and the perfusion velocity was controlled by the drip infusion with collagenase perfusate containing 10% of fetal calf serum. Results?Total success rate of catheterization in the improved group was as high as 95%, and the success rate of first attempt in the improved group was significantly higher than that in the traditional group (94.7% vs. 68.8% ). Liver perfusion in the improved group was symmetrical with the high yield of hepatocytes up to 1.07×10⁶ per gram of mouse weight and 92.16% of average cell vitality, which were higher than those in the traditional group. Conclusion?The retrograde liver perfusion through the heart is a simple and easy-to-learn method to isolate mouse primary hepatocytes, which also could guarantee the satisfactory yield and vitality of primary hepatocytes.