Background: A high unmet need remains for first-line cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma (la/mUC).Immune checkpoint inhibitors have been implemented in the management of cisplatin-ineligible la/mUC. Penpulimab is a programmed death 1 (PD-1) inhibitor which had approved by the Chinese National Medical Products Administration (NMPA) on August 2021. Anlotinib is a novel oral multi-target tyrosine kinase inhibitor and primary targeted to VEGFR, FGFR, PDGFR and c-Kit. The current prospective single-arm phase Ⅱ clinical trial (ChiCTR1900028022) aimed to assess the efficacy and safety of penpulimab plus anlotinib as first-line therapy for la/mUC. Methods: Patients (pts) who had no prior systemic therapy and at least one measurable lesion according to RECIST v1.1 with histologically or cytologically confirmed locally advanced unresectable or metastatic urothelial carcinoma (including unresectable or metastatic urothelial carcinoma of the bladder, urethra, ureter, or renal pelvis), 18-75 years, ECOG 0-2, were considered eligible for enrollment. Patients received 200 mg penpulimab intravenously on day 1 plus anlotinib, 8 mg oral daily for days 1-14 of a 21-day cycle until confirmed disease progression, intolerable toxicity, physician/patient decision to withdraw, or completion of 24 months of treatment. The primary endpoint was objective response rate (ORR), and the secondary endpoints included disease control rate (DCR), progression-free survival (PFS) and safety. Results: As of Dec. 2021, 10 pts were enrolled, 9 pts had the best overall response assessments which inferred the ORR of 66.7%（95% CI, 29.9-92.5）and the DCR of 88.9% (PR in 6 pts and SD in 2 pts; 95% CI, 51.8-99.7). The median PFS was 8.2 months（(95% CI, 8.0-8.4). Any grades of adverse events (AEs) were observed in 90% (9/10) of pts, containing hyperthyroidism (33%), Creatinine increased (33%),haematuria (22%), anaemia (22%), hypertension (11%), fatigue (11%). High grade AEs of grade Ⅳ liver dysfunction were observed in one patient (11%). Conclusions: An effective treatment strategy for cisplatin-ineligible patients and for patients unable to receive chemotherapy is an important unmet clinical need. Penpulimab in combination with anlotinib in pts with la/mUC showed encouraging efficacy and satisfactory safety. Clinical trial information: ChiCTR1900028022.