To assess the antitumor activity and safety of tipifarnib, a highly potent and selective farnesyltransferase inhibitor, we performed a phase II clinical trial in patients with advanced and refractory urothelial carcinoma harboring missense mutations. A total of 245 adult patients with previously treated, advanced urothelial carcinoma entered the molecular screening program including . Those with missense mutations or :rs2075606 received oral tipifarnib 900 mg twice daily on days 1-7 and 15-21 of 28-day treatment cycles. The primary endpoint was progression-free survival at 6 months (PFS6). We identified 16 (7%) missense mutations (G13R, 7; Q61R, 4; G12S, 3; G12C, 2) and 104 (46%) :rs2075606 carriers. In 21 patients enrolled in the study, 14 and 7 patients had missense mutations and :rs2075606, respectively. The most frequently observed adverse events included fatigue (86%) and hematologic toxicities. With a median follow-up of 28 months, 4 patients (19%) reached PFS6: 3 had missense mutations and one patient, enrolled as an carrier, had frameshift insertions at H27fs and H28fs rendering a nonsense mutation. The overall response rate by intent-to-treat analysis was 24% (4 missense and one nonsense frameshift HRAS mutation); no response was observed in patients with urothelial carcinoma with wild-type tumors. Five responses were observed in 12 evaluable patients of 15 with tumors carrying mutations. Oral tipifarnib resulted in a manageable safety profile and encouraging antitumor efficacy against treatment-refractory urothelial carcinoma containing mutations.