Background: Glioblastoma (GBM) is the most common primary malignant brain tumor and one of the most deadly forms of cancer in humans. Given the temozolomide’s minimal benefit in O⁶-methylguanine-DNA Methyltransferase (MGMT) unmethylated GBM, new and effective therapies are urgently needed. In this study, we aimed to assess the safety and efficacy of anlotinib, a novel multitarget tyrosine kinase inhibitor for VEGFR, PDGFR, and c-Kit, in patients with MGMT unmethylated GBM. Methods: This was an open-label, single -center, single-arm, phase 2 trial. Eligible patients included those aged 18 to 70 years who had newly diagnosed histologically confirmed GBM with unmethylated MGMT promoter region. The cutoff value ≥ 10% was used to classify MGMT methylated vs. unmethylated cases. After initial surgical resection, patients with GBM received concurrent radiotherapy (2.0 grays [Gy]/day; total dose, 60 Gy) and anlotinib (beginning on day 43 after surgery, at a dose of 12 mg orally once daily on days 1-14 of a 21-day cycle), followed by 6 cycles of maintenance therapy with anlotinib (at a dose of 12 mg orally once daily on days 1-14 of a 21-day cycle). Patients were re-evaluated every six weeks. The primary endpoint was progression-free survival (PFS), which is defined as the interval from the initiation of anlotinib treatment until the date of disease progression or death. The secondary endpoints were overall survival (OS) and safety. Standard Response Assessment in Neuro-Oncology (RANO) criteria were used to determine tumor response and progression. Adverse events were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0. This trial was registered with chictr.org.cn, ChiCTR2000040116. Results: A total of 32 participants were enrolled from September 2020 to June 2022. All patients were with IDH wild-type and MGMT unmethylated status. The median age was 50 years (range, 27-65), and 19 (59.4%) were male. The median follow-up duration was 14.8 months (range, 5.5-23.7). The median PFS was 10.5 months (95%CI: 8.5-12.5), and the median OS was not reached. Treatment-related adverse events (TRAEs) occurred in 13 (40.6%) patients of patients and grade 3 adverse events were observed in 4 (12.5%) patients. The most common toxicities were hypertension (13 [41%]), hand-foot syndrome (12 [38%]), hematuresis (9 [28%]) and liver dysfunction (5 [16%]). Grade 3 therapy-related adverse events included liver dysfunction (2 [6.3%]), hypertension (1 [3.1%]), hand-foot syndrome (1 [3.1%]). No grade 4-5 or unexpected adverse events were observed. Conclusions: This preliminary analysis suggested that anlotinib had promising efficacy and favorable tolerance as treatment of newly diagnosed GBM with unmethylated MGMT promoter. Clinical trial information: ChiCTR2000040116.