Background: Raltitrexed-based chemotherapy regimen is one of the common regimens for the treatment of metastatic colorectal cancer (mCRC). This prospective observational real-world study aimed to evaluate the safety and effectiveness of raltitrexed administered to Chinese patients with mCRC in real life setting. Methods: This is a prospective, multicenter, real-world study. Prospectively registered Patients received second-line treatment of raltitrexed plus irinotecan combined with or without target therapy until progression disease or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were objective response rate (ORR), disease control rate (DCR), overall survival (OS), quality of life (QOL) and safety. Totally,1000 patients were required for primary point testing. Results: Between May 2018 and December 2021,a total of 1039 patients from 57 centers were screened for enrollment,among which 271 patients were treated with raltitrexed plus irinotecan, 753 patients accepted target therapy (bevacizumab or cetuximab) additionally, and 15 patients combined with other drugs. Overall mPFS was 6.8 months (95%CI: 6.5-7.1),ORR was 20.2%, and DCR was 85.7%; The ORR of combined with or without target therapy were 21.6% and 16.2% (p = 0.038),respectively, the DCR were 88.2% and 79.0% (p < 0.001). The mPFS of combined without or with target therapy were 5.2 months (95% CI: 4.7 to 5.7) and 7.3 months respectively (95% CI: 7.0-7.7) [HR = 0.67, 95% CI 0.56̃0.80, p < 0.001], The mPFS of combined with bevacizumab or cetuximab were 7.4 months (95% CI: 7.0 -7.8) and 6.8 months (95% CI: 5.9-7.7) [HR = 1.15, 95% CI 0.88̃1.51, p = 0.3]. mOS has not yet reached. Majority of treatment-related adverse events (TEAEs) were grade I or II. The most common grade III or IV TRAEs reported by 116 patients (11.2%) were aspartate aminotransferase increased (4.0%), alanine aminotransferase increased (3.7%), neutrocytopenia (2.7%), glutamyltransferase increased (2.5%), leukocytopenia (1.1%). Conclusions: The real-world study confirmd that raltitrexed was an effective and safe regimen for the second-line treatment in Chinese patients with mCRC, especially combined with target therapy additionally, which was aligned with previous trials. Clinical trial information: ChiCTR1800016185.