Abstract Purpose Patients with locally advanced rectal cancer (LARC) who underwent total neoadjuvant therapy (TNT) showed an increase in the percentage of complete pathological response (pCR). The purpose of this study was to determine the correlation between Ki-67, tumor-infiltrating lymphocytes (TIL), and TNT in LARC patients. Method In total, one hundred fifty-nine patients with LARC were included in this prospective study. The international working group was used to categorize the TIL into three groups based on the percentage and density of staining: group 0 (0–10%), group 1 (11–59%), and group 2 (≥ 60%). Ki-67 expression was classified as low (≤ 50%) or high (> 50%). Result Most patients had tumor grade III (74.2%) and T2–T3 (78.6%). Lymph node involvement (48.7%) and tumor size ≥ 3 cm were detected in approximately half of the patients. Forty-four percent of patients had a high Ki-67 index; 15.7% of patients belonged to group 1, and 21.4% belonged to group 2. pCR was detected in 18.2% of the patients. TIL and Ki-67 levels were significantly correlated with pCR (p = 0.001 and 0.003 for multivariate analysis and 0.001 and 0.03 for univariate analysis, respectively). Conclusion There was a statistically significant correlation between Ki-67, TIL, and pCR following TNT protocol, which may help maximize the therapeutic outcome.