BACKGROUND: Fluorescence-guided lymphadenectomy (FLND) using indocyanine green (ICG) has emerged as a promising technique to enhance the accuracy of lymphadenectomy in rectal cancer surgery. Effective lymphadenectomy is crucial for improving prognosis in patients with advanced rectal cancer, but it remains technically challenging and controversial. METHODS: This prospective nonrandomized controlled study was conducted involving 129 patients underwent laparoscopic surgery, and 64 patients assisted by FLND. Patients received submucosal ICG injections before surgery to facilitate FLND. Lymph nodes were categorized as station 251, station 252, or station 253 based on their anatomical locations. The effectiveness of FLND was evaluated by comparing the number of harvested and metastatic lymph nodes between the FLND and control groups. RESULTS: The FLND group demonstrated a significantly higher median number of harvested station 253 lymph nodes compared to the control group (2.0 vs. 1.0, P = 0.007). The FLND cohort had a shorter postoperative hospital stay (6 days vs. 8 days, P <
0.001) and similar rates of postoperative complications compared to the control cohort. The study found no significant differences in the median number of harvested station 251 (10.0 vs. 11.0, P = 0.872) and station 252 (6.0 vs. 5.0, P = 0.369) lymph nodes between the groups. Univariate and multivariate analyses indicated that FLND significantly increased the harvested lymph node count. CONCLUSION: Radical surgery assisted by FLND significantly improves the accuracy and yield of lymphadenectomy in mid-low rectal cancer, enhancing surgical outcomes and patient prognosis. Future advancements in fluorescence imaging and related technologies hold promise for further improving the clinical effectiveness of this technique.