PURPOSE: Total neoadjuvant therapy (TNT) has emerged as a therapeutic approach for locally advanced rectal cancer (LARC). However, the optimal chemotherapy cycles within TNT remain uncertain. This study aimed to evaluate and compare the prognostic efficacy of varying cycles of chemotherapy during TNT for LARC. METHODS: Patients diagnosed with LARC (T3-4N0M0/T1-4N1-2M0), who underwent TNT or chemoradiotherapy followed by total mesorectal excision (TME) between 2015 and 2020, were retrospective included. Patients were categorized into three groups based on their neoadjuvant strategy: CRT (long-course chemoradiotherapy), STNT (long-course CRT with one to three cycles of chemotherapy), and LTNT (long-course CRT with four or more cycles of chemotherapy). Propensity score matching (PSM) based on gender, age, body mass index, tumor distance from the anal verge, clinical T stage, clinical N stage, and mesorectal fascia status was employed to reduce confounding bias. Primary endpoints were disease-free survival (DFS) and metastasis-free survival (MFS). RESULTS: The study comprised 372 patients, with 73 patients in each group after PSM. Compared with CRT, both STNT and LTNT demonstrated improved DFS (5-year rate: 59.7% vs. 77.8% vs. 76.5%, p = 0.027) and MFS (5-year rate: 65.1% vs. 81.3% vs. 81.4%, p = 0.030). There was no difference in DFS or MFS between STNT and LTNT. These favorable outcomes were consistent among subgroups defined by tumor distance from the anal verge ≥ 5 cm, clinical T3 stage, clinical N positive status, or involved mesorectal fascia. CONCLUSION: Compared to CRT, both STNT and LTNT demonstrated improved DFS and MFS outcomes. Notably, survival outcomes were similar between STNT and LTNT, suggesting that chemotherapy cycles in TNT may not significantly impact survival.