Background/Aims: Recent reports have demonstrated that some patients are unable to undergo scheduled liver resection after preoperative portal vein embolization due to insufficient hypertrophy of the future remnant liver. The present study examined whether two-stage portal vein ligation (PVL) increases hypertrophy of the future remnant liver compared to conventional PVL in rats. Methods: Rats were divided into 3 groups: group A, ligation of left primary branch; group B, ligation of right and left primary branches; group C, ligation of the left primary branch, followed by 2-stage PVL 7 days postoperatively. To evaluate liver regeneration, the proliferating cell nuclear antigen labeling index (LI), mitotic index (MI) in the caudate lobe and weight ratio of caudate lobe to body weight were measured. Results: The weight ratio of caudate lobe to body weight was significantly higher in group C than in groups A or B 14 days postoperatively. In groups A and B, LI and MI in the caudate lobe peaked 2 days postoperatively, then decreased to preoperative levels by 7–8 days postoperatively, but remained significantly elevated until 10–14 days postoperatively in group C. Conclusion: Two-stage PVL increases hypertrophy of the future remnant liver compared to conventional PVL in rats.