Background: Radical surgery involving cystectomy and nephroureterectomy is the standard of care for patients with localized muscle-invasive urothelial carcinoma (MIUC). In recent years, neoadjuvant- and adjuvant-containing multimodality therapies have gradually been adopted in attempt to eradicate micrometastatic disease and improve the outcomes achieved with radical surgery alone. This study aims to evaluate the efficacy and safety of anlotinib (a multi-target tyrosine kinase inhibitor of angiogenesis) with gemcitabine and cisplatin or anlotinib with penpulimab (a humanized anti-PD-1 IgG1 monoclonal antibody) as neoadjuvant and adjuvant therapy in patients with MIUC. Methods: In this open-label, prospective, phase 2 study, eligible patients include those with histologically confirmed clinical stage T1-T4N1-3M0, T3-4N0M0 MIUC (tumors of the bladder, renal pelvis, ureter and urethra) fit and planned for radical cystectomy, nephroureterectomy or ureterectomy. Main exclusion criteria are distant metastatic disease; prior chemotherapy, anti-angiogenic therapy, radiation therapy or immunotherapies; active autoimmune disease; uncontrolled hypertension or patients at high bleeding risk. Patients will be enrolled and receive treatment based on PD-L1 status in tumor tissue. The PD-L1 status of eligible patients will be centralized and assessed using an anti-PD-L1 antibody and an immunohistochemical (IHC) assay. PD-L1 positivity was defined as combined positive score (CPS) ≥1 (previously reported as and equivalent to CPS ≥1%). The study treatment is stratified into two cohorts based on PD-L1 expression levels. In cohort 1, the PD-L1 negative patients with MIUC will receive neoadjuvant anlotinib (12 mg orally QD D1-14 every 3 weeks) + gemcitabine (1000 mg/m², intravenously [IV] on days 1 and 8) + cisplatin (35 mg/m² IV on days 1 and 8) for a maximum of 4 cycles followed by radical surgery and 2 cycles of adjuvant treatment post-surgery. In cohort 2, PD-L1 positive patients will receive anlotinib (12 mg orally QD D1-14 every 3 weeks) + penpulimab (200 mg IV every 3 weeks) for a maximum of 4 cycles followed by radical surgery and 2 cycles of adjuvant treatment post-surgery. After the completion or discontinuation of the study treatment, safety follow-up and survival follow-up will be performed. Tumor imaging was performed by computed tomography (preferred) or magnetic resonance imaging at baseline, and every 6 weeks during treatment, then every 6 months through year 2. Objective response rate (ORR) is the primary endpoint. Secondary endpoints included pathological complete response rate (pCR), disease-free survival (DFS), overall survival (OS) and safety. Recruitment have begun in October 2022. Currently, the study is active with 1 participant has been enrolled. Clinical trial information: ChiCTR2200063085.