Background: Biliary tract cancer (BTC) is a rare but aggressive disease, and most patients present with unresectable or metastatic disease. Gemcitabine combined with cisplatin (GP) has been established as the standard first-line treatment in patients with unresectable BTC. Nevertheless, no standard second or further-line treatment regimen has been approved in this disease. Fruquintinib is an oral multi-kinase inhibitor targeting vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2 and VEGFR3. In this single arm phase II study, we explored the efficacy and safety of fruquintinib as a novel second or further-line therapy in advanced or metastatic BTC. Methods: BTC patients with ECOG PS 0-1 and adequate liver, kidney and bone marrow function were given fruquintinib (5 mg, PO, D1-21, Q4W) until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS), and the secondary endpoints included objective response rate (ORR), disease control rate (DCR), overall survival (OS), and safety. The exploratory endpoint was the correlation of clinical outcomes with biomarkers. Results: 20 patients were enrolled in the study. 9(45.0%) were men, and the median age was 57.5(36-78) years. 18 patients were diagnosed with ICC, 1 with ECC, and 1 with GBC. 6 (30%) patients were refractory to second-line treatment, and 5 (25%) patients were refractory to third-line treatment. 18 patients were assessable for response. The median PFS was 4.6 months (95%CI: 2.1-7.1 months). ORR was 5.6%, and DCR was 66.7%. The incidence of treatment-related grade 1-2 adverse events (TRAEs) was 83.3%, and the most common grade 1-2 AEs were hand-foot syndrome (66.6%), fatigue (61.1%), stomatitis (44.4%), and hypertension (38.9%). Conclusions: Fruquintinib showed promising efficacy and acceptable toxicity as second or further-line therapy in advanced or metastatic BTC. Clinical trial information: NCT04156958.