Objective To investigate the effect of meticulousness of pathologists on the lymph node harvest after radical resection of invasive rectal carcinoma in paients following neoadjuvant chemoradiotherapy. Methods The clinical data of 191 patients with rectal cancer (stage Ⅰ to Ⅲ) undergoing radical excision after neoadjuvant chemoradiotherapy in Department of Colorectal Surgery, Changhai Hospital, Second Military Medical University from Jan. 2005 to Dec. 2014 were retrospectively analyzed. Since Jan. 2009 when multidisciplinary cooperation was carried out, pathologists and surgeons suggested that no less than 15 lymph nodes in single rectal specimen should be found by meticulousness of pathologists, according to which patients were divided into routine sampling group (n=46) and meticulousness group (n=145). The harvest number and positive rate of postoperative lymph nodes were compared between the two groups. Results The lymph node retrieval in the meticulousness group was significantly more than that in the routine sampling group (13.1±4.9 vs 9.6±4.0, P<0.01), and the percentage of patients with the lymph node harvest equaling or more than 12 was significantly increased (69.7% vs 33.3%, P<0.01). There were no significant differences in the positive node harvest number (1.8±2.9 vs 1.9±3.8, P=0.334) or positive rate of lymph nodes (50.0% vs 38.6%, P=0.172) between the two groups. The positive rate of lymph nodes of patients in T0, T1, T2, T3, and T4 stages in the two groups were 25.0%(1/4) and 22.7%(5/22), 0.0%(0/1) and 33.3% (1/3), 9.1%(1/11) and 25.0%(8/32), 70.0%(21/30) and 47.1%(40/85), and 0.0%(0/0) and 66.7%(2/3), respectively. There were no significant differences in T stages between the two groups (χ²=4.55, P=0.209). Conclusion The lymph node harvest number is increased by meticulousness of pathologists after multidisciplinary cooperation, while the positive node harvest number and positive rate of lymph nodes are not increased, suggesting that supplementary measures should be taken to obtain more accurate lymph node status of patients with rectal cancer after neoadjuvant chemoradiotherapy.