Purpose: Increased activity of STAT3 is associated with progression of head and neck squamous cell carcinoma (HNSCC). Upstream activators of STAT3, such as JAKs, represent potential targets for therapy of solid tumors, including HNSCC. In this study we investigated the anti-cancer effects of ruxolitinib, a clinical JAK1/2 inhibitor, in HNSCC preclinical models including patient-derived xenografts (PDXs) from patients treated on a window-of-opportunity trial. Methods: HNSCC cell lines were treated with ruxolitinib, and the impact on activated STAT3 levels, cell growth, and colony formation was assessed. PDXs were generated from HNSCC patients who received a brief course of neoadjuvant ruxolitinib on a clinical trial. The impact of ruxolitinib on tumor growth and STAT3 activation was assessed. Results: Ruxolitinib inhibited STAT3 activation, cellular growth and colony formation of HNSCC cell lines. Ruxolitinib treatment of mice bearing a HNSCC cell line-derived xenograft significantly inhibited tumor growth compared with vehicle-treated controls. The response of HNSCC PDXs derived from patients on the clinical trial mirrored the responses seen in the neoadjuvant setting. Baseline active STAT3 (pSTAT3) and total STAT3 levels were lower, and ruxolitinib inhibited STAT3 activation in a PDX from a patient whose disease was stable on ruxolitinib, compared to a PDX from a patient whose disease progressed on ruxolitinib and where ruxolitinib treatment had minimal impact on STAT3 activation. Conclusions: Ruxolitinib exhibits antitumor effects in HNSCC preclinical models. Baseline pSTAT3 or total STAT3 levels in the tumor may serve as predictive biomarkers to identify patients most likely to respond to ruxolitinib.