Background: Gemcitabine combined with cisplatin (GP) has been established as the standard first-line treatment in patients with unresectable biliary tract cancers (BTC), a heterogeneous group of rare and aggressive malignancies arising from the epithelium of the biliary duct system. Nevertheless, there has been no approved standard of care for second-line treatment. In this study, we explored the efficacy and safety of the combination of anlotinib and sintlimab as a novel second-line regimen in BTC. Methods: In this phase II trial, patients with advanced BCT, who experienced progression after first-line chemotherapy, received anlotinib (12 mg, PO, D1-14, Q3W) combined with sintlimab (200 mg, iv, D1, Q3W) until disease progression or unacceptable toxicity. The primary endpoint was OS, and the secondary endpoints included ORR, PFS, DCR, and safety. The exploratory endpoint was the correlation of clinical outcomes with biomarkers. Results: From Aug 2019 to Sep 2020, 17 patients were enrolled. 9(52.9%) were men, and the median age was 59(43-69) years. Nine patients were diagnosed with ICC, 2 with ECC, and 6 with GBC. 15 patients were assessable for response. At a median follow-up of 8.76 months (95%CI: 3.60-13.94 months), the primary endpoint OS has not been reached, the median PFS was 6.50 months (95%CI: 3.60-9.40 months). ORR was 40.00%, and DCR was 86.67%. The incidence of treatment-related adverse events (TRAEs) was 70.60%, and the most common grade 1-2 AEs were hypertension (70.60%), diarrhea (17.65%), and hypothyroidism (17.65%). Conclusions: Anlotinib plus sintlimab demonstrates significant clinical activity and acceptable toxicity in patients with advanced BTC who failed first-line chemotherapy. Clinical trial information: ChiCTR1900022003.