There is no regulation in the current guidelines on the sequence of ligation of the inferior mesenteric artery and vein during rectal cancer surgery owing to a lack of sufficient evidence. Circulating tumor cells (CTCs) in peripheral blood can be used as potential indicators for predicting prognosis in colorectal cancer patients. This study aims to explore the feasibility of different ligation sequences for the inferior mesenteric vessels and their potential influence on CTCs.This pilot study involved 29 stage I-III rectal adenocarcinoma patients undergoing laparoscopic surgery. Patients were allocated into two groups based on the sequence of vascular ligation: vein-first (V-first) and artery-first (A-first). The primary objective was to assess the impact of the ligation sequence on peripheral blood CTC levels pre- and post-operatively. Secondary outcomes included intraoperative complications, surgical duration, blood loss, and number of lymph nodes harvested, and postoperative complications. The study was approved by the ethics committee of our hospital (SCCHEC-02-2024-102), and all patients provided informed consent.No significant differences were found between the two groups regarding surgical duration, blood loss, lymph nodes harvested, or postoperative complications. A reduction in CTCs postoperatively was observed in 36% of patients in the V-first group, in comparison to 20% in the A-first group.Both A-first and V-first ligation sequences are viable and safe options in laparoscopic rectal cancer surgery. The V-first approach may be more effective in reducing levels of CTCs in peripheral blood. Further randomized studies are warranted to explore these findings comprehensively.© 2024. The Author(s).