We aimed to assess the clinical efficacy of glutaraldehyde-fixed human umbilical vein endothelial cell (HUVEC) vaccine for the treatment of patients with recurrent glioblastoma. Patients of a HUVEC vaccine group received intradermal injections of 5 × 107 HUVEC weekly during the first month, and every 2 weeks from the second month, until progression of the disease was observed. Salvage treatment consisted of multimodal chemotherapy, radiation, including gamma-knife therapy, and/or repeated surgery, when feasible. Hazard ratios for death were calculated using a Cox model. A total of 17 patients with recurrent glioblastoma were enrolled in this study. All the patients received the initial treatment consisting of maximal safe surgical resection, followed by radiotherapy of 50–80 Gy or more, with concomitant and adjuvant chemotherapy consisting of temozolomide or nimustine (ACNU). A total of 352 vaccinations were performed for the patients of the HUVEC vaccine group (median number of vaccination = 11 doses; range 3–122 doses). The median progression-free survival and overall survival were 5.5 and 11.4 months, respectively. The median overall survival from the diagnosis was 24.3 months. The HUVEC vaccine therapy significantly prolonged the tumor doubling time and contributed to reducing the tumor growth rate. Hematological adverse reactions due to chemotherapy were recognized: one patient experienced grade III leukocytopenia and one showed grade II lymphocytopenia. Associated with the HUVEC vaccine therapy, a delayed-type hypersensitivity-like skin reaction developed at the injection site. The HUVEC vaccine therapy effectively controlled disease progression, without evident adverse effects, except for a delayed-type hypersensitivity-like skin reaction at the injection site.