Background: UC is a common malignancy with poor outcomes in patients with metastatic disease. The majority of urothelial cancers harbor alterations in key retinoblastoma (Rb) pathway genes with CDKN2A alterations in approximately 35% of tumors, leading to loss of Rb tumor suppressor function with subsequent cell cycle progression and unchecked cell proliferation. Palbociclib is an oral, selective inhibitor of CDK4/6 that prevents Rb phosphorylation to promote cell cycle arrest. Methods: In this phase II trial, biomarker-selected (p16 loss and intact Rb by tumor immunohistochemistry [IHC]) patients with metastatic platinum-refractory UC received palbociclib 125mg po daily for 21 days of a 28-day cycle. The primary endpoint was progression-free survival at 4 months (PFS4) using a Simon’s two-stage design. Prespecified tumor analysis with next generation sequencing (NGS) including Rb pathway alterations was conducted. Results: 12 patients (67% male, median age 68 years) were enrolled. Post-platinum prognostic factors included hemoglobin < 10 g/dL, 17%; liver metastases, 0%; median time from prior therapy, 5.0 months; and Eastern Cooperative Oncology Group performance status < = 1, 92%. Overall, two of 12 patients (17%) achieved PFS4 with insufficient activity to advance to stage 2. 58% of patients had grade ≥3 hematologic toxicity with 25% grade 3 anemia and 17% grade 3 thrombocytopenia. Using NGS, the most frequently observed somatic mutations were ARID1A, MLL2, PIK3CA, and TP53 (55% for each). No patients had CDKN2A alterations, although the majority (82%) of patients had a cell cycle pathway alteration (CDKN1A 27%, CDKN2B 27%, E2F3 18%). There was no correlation between genomic alterations and clinical outcome. Conclusions: Palbociclib demonstrated limited activity in patients with platinum-refractory metastatic UC selected for p16 loss and intact Rb by IHC. Further development of palbociclib should only be considered with improved integral biomarker selection such as NGS or in rational combination with other therapies. Clinical trial information: NCT02334527