Background: Dysregulation of the JAK/STAT pathway in newly-diagnosed high-grade gliomas (nHGG) is linked to enhanced survival and proliferation of tumor cells. Ruxolitinib, a small molecule inhibitor of JAK1, JAK2, and JAK3, limits glioma growth in preclinical models. This concept was explored in a phase I trial (NCT03514069), whose final report on toxicity is presented here. Methods: This non-randomized prospective study included 60 WHO Grade 3-4 nHGG patients who received standard of care (SOC) therapy along with ruxolitinib in a 3+3 dose-escalation design; level 1 of 10 mg BID, level 2 of 15 mg BID, level 3 of 20 mg BID, level -1 of 5 mg BID. The primary study objective was the determination of the maximum tolerated dose (MTD) of ruxolitinib in combination with chemoradiation. The secondary objective was the determination of safety, overall survival (OS), and progression-free survival (PFS). The exploratory aims were to investigate relationships between clinical outcomes and genomic signatures. Results: 60 patients were enrolled, with a median age of 60.5 years (range 22-78). 23 (38%) patients were female and 37 (62%) were male. 29 (48%) were MGMT unmethylated and received ruxolitinib with radiation of 60 Gy over 6 weeks. 31 (52%) patients were MGMT methylated and received ruxolitinib with 75 mg/m² of temozolomide (TMZ) with radiation of 60 Gy over 6 weeks. The 1-year OS rate was 77% for all GBM patients; 62% for arm 1 (unmethylated MGMT) and 93% for arm 2 (methylated MGMT). Median OS for arm 1 was 18.1 months (10.1, NA) and was not reached for arm 2. MTD for both cohorts was 20 mg BID. No dose-limiting toxicities were observed. Toxicities attributable to study medications included four grade 4 AEs including seizure, respiratory distress, somnolence, and thromboembolic event along with 14 grade 3 adverse events (AEs), including respiratory distress, seizure, gait disturbance, weakness, thrombocytopenia, cognitive disturbance, urinary retention, and meningitis. Conclusions: Ruxolitinib therapy is safe and feasible in combination with TMZ and radiation. Efficacy of ruxolitinib plus SOC appears promising compared to historical benchmarks for both MGMT methylated and MGMT unmethylated cohorts. A randomized phase 2 trial is planned. Clinical trial information: NCT03514069.