Aims This study aimed to investigate the capability of methylated SEPT9 (mSEPT9) assay in assessing the neoadjuvant therapy (NAT) response,the minimal residual disease (MRD) and predicting the recurrence of stage III colorectal cancer (CRC). Materials & methods We prospectively recruited 124 stage III CRC patients. All patients received pre-surgical NAT and subsequent curative surgery, and were followed up to 627 days. Blood samples were collected before NAT, after NAT and after surgery. Measurements of mSEPT9 and CEA were performed. Results mSEPT9 exhibited a positive detection rate (PDR) of 82.6% for stage III CRC, significantly higher than that of the carcinoembryonic antigen (CEA) (60.3%,p < 0.001). The levels of mSEPT9 and CEA significantly decreased after NAT. This can be observed for patients with complete response (CR), partial response (PR), stable disease (SD) and tumor regression grade (TRG) 1-3 in mSEPT9 and patients with PR, SD and TRG 2-3 in CEA. Stepwise percentage decrease of marker levels was more prominent in mSEPT9 than in CEA following NAT and surgical treatment. Kaplan-Meier analysis suggested that mSEPT9 significantly stratified the patient recurrence-free survival(RFS) before NAT, after NAT and after surgery. In contrast, CEA significantly stratified RFS after NAT and after surgery, while CEA levels before NAT did not significantly stratify RFS. Conclusion mSEPT9 exhibits better applicability than CEA in assessment in terms of patient coverage, sensitivity and the pre-NAT recurrence prediction.