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This study aimed to investigate the application of fat clearance in cases of rectal cancer after neoadjuvant chemoradiation.All patients who underwent proctectomy (R0 resection) from 1998 to 2007 were included. N1 and N2 stages were regarded as N+ stage.Two hundred thirty-seven patients were identified, including 157 patients in the neoadjuvant group and 80 patients in the nonneoadjuvant group. In both groups, patients were assigned to receive the traditional method of harvesting lymph nodes, or the fat clearance method. Before July 2001, the patients received the traditional method, and after July 2001, they received exclusively the fat clearance method. In the nonneoadjuvant group, there was no significant difference in the number of positive lymph nodes (0.5 ± 0.2 vs. 1.0 ± 0.3, P = 0.235), N stage (P = 0.265), or patients with N+ stage (7/31 vs. 16/49, P = 0.332) between the two methods, even though the total lymph node harvest was significantly increased by use of the fat clearance method (9.6 ± 1.3 vs. 27.6 ± 2.5, P < 0.001). In contrast, the total lymph node retrieval (5.2 ± 0.6 vs. 20.4 ± 1.2, P < 0.001), number of positive lymph nodes (0.4 ± 0.2 vs. 1.2 ± 0.3, P = 0.007), N stage (P = 0.005), and patients with N+ stage (6/51 vs. 34/106, P = 0.006) were all increased by fat clearance in the neoadjuvant group. Moreover, the number of patients with N+ stage was stratified by T stage level (T0–T4) to eliminate the background bias, and the results were confirmed.The utilization of the fat clearance technique significantly influences lymph node staging in patients with rectal cancer following neoadjuvant chemoradiation. These findings suggest that fat clearance may represent a useful tool in all patients receiving neoadjuvant therapy; a more generalized application in colorectal carcinoma specimens remains controversial and warrants further investigation.