Background: Glioblastoma (GBM) is a highly vascularized tumor and there are few treatment options after disease recurrence. This study assessed the efficacy and safety of anlotinib, a multitarget tyrosine kinase inhibitor, combined with temozolomide in the treatment of these patients. Methods: This is an open-label, single-arm, phase II trial (ChiCTR2000028957). Patients with histologically confirmed GBM with definite progression after surgery followed by radiotherapy and temozolomide chemotherapy were eligible for inclusion. Additional inclusion criteria were 1) 18-70 years old, 2) KPS≥60, 3) disease progression on MRI as defined by Response Assessment in Neuro-Oncology (RANO) criteria at least 12 weeks (wks) after completion of radiotherapy，4) antiangiogenic therapy naïve. All patients received temozolomide (150-200mg/m², orally, QD, d1-5/4wks) and anlotinib (10mg, orally, QD, d1-14/3wks) until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR, defined as partial and complete responses according to RANO criteria). Safety assessment was done in patients who received at least one dose of study agent. Results: Twenty-one patients (9 males and 12 females) were enrolled between May 2020 and July 2021, and the median age is 55 (range 27-68) years old. Molecular pathological types included MGMT promoter methylated (n = 5), IDH mutation (n = 5), 1p/19q codeletion (n = 8), and TERT promoter mutation (n = 12). Analyses included data collected through December 15, 2021. Tumor response occurred in 17 patients, of which 9 patients had a complete response, and ORR was 81.0% (17/21). There were also 3 patients achieved stable disease and the disease control rate was 95.2% (20/21). Median progression-free survival (PFS) was 13.2 months [95%CI 3.90-22.52], and the PFS at 6 months was 60.0%. The incidence of adverse events of grade 3 was 28.6%, with a higher incidence of elevated AST (25.0%) and ALT (20.0%), hypertension (16.7%), leukopenia (15.4%). No grade 4 or treatment related death occurred in this study through the follow-up. Overall, toxicities are mild and manageable. Conclusions: Anlotinib combined with temozolomide is efficacious and well-tolerated in recurrent GBM patients. Clinical trial information: ChiCTR2000028957.