Background: Human epidermal receptor growth factor 2 (HER2) -expressing or amplified has been identified in 2-6% of patients with stage 3/4 colorectal cancer (CRC), and is associated with poor prognosis, however there are currently no approved HER2-targeted therapies for CRC in China. Herein, we aimed to study the antitumor activity and safety of Disitamab vedotin (RC48), a novel humanized anti-HER2 antibody conjugated to MMAE via a cleavable linker, combined with fruquintinib in patients with HER2-expressing or -amplified metastatic colorectal cancer. Methods: In this prospective, exploratory, single-arm study, patients diagnosed pathologically with mCRC, 18-75 years old harboring HER2 expression or HER2 mutation/amplification, and received at least two lines of treatment. HER2 expression was defined as a score of 1+, 2+ or 3+ on immunohistochemical [IHC] analysis, or mutation/amplification by NGS. Patients received RC48 2.5mg/kg intravenously every 2 weeks. Meanwhile, fruquintinib 3mg was administered orally once daily on days until disease progression, death, intolerable toxicity, withdrawal of consent. The primary endpoint was objective response rate (ORR) per RECIST v1.1; the secondary endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Results: A total of 12 patients were enrolled from November 25, 2022 to June 6, 2023. The results of baseline molecular profiling revealed 4 with IHC 1+, 4 with IHC 2+, 2 with IHC 3+, and 2 with HER2 amplification. In the metastatic setting, patients had received a median of 4 (range, 3-6) lines of prior systemic therapy. In the per-protocol set, the investigator-assessed ORR was 18.2% (2/11) and DCR was 90.9% (10/11). Median PFS was 5.5 months (95% CI: 2.7-8.2) and the median OS was 12.3 months (95% CI: 5.3-19.3). During the study process, 11 (91.7%) patients experienced adverse events (AEs), among which, 4 (33.3%) experienced grade 3 treatment-related AEs (TRAEs). The most common grade 3 TRAEs were neutropenia (25%), leukopenia (16.7%), and hypertension (8.3%). Moreover, no grade 4 or 5 TRAEs were observed. In addition, 4 (33.3%) patients experienced a dose reduction. No patients experienced dose interruption or discontinuation due to TRAEs. Conclusions: RC48 combined with fruquintinib has shown promising efficacy and a manageable safety profile for HER2-expressing or -amplified mCRC patients refractory to at least two standard treatment failures. Clinical trial information: NCT05661357.