This multicenter clinical study was performed to assess the safety and effectiveness of Trinity Evolution® (TE), a viable cellular bone allograft, in combination with a PEEK interbody spacer and supplemental anterior fixation in patients undergoing anterior cervical discectomy and fusion (ACDF). In a prospective, multi-center study, 31 patients that presented with symptomatic cervical degeneration at one vertebral level underwent ACDF with a PEEK interbody spacer (Orthofix, Inc., Lewisville, TX, USA) and supplemental anterior fixation. In addition all patients had the bone graft substitute, Trinity Evolution (Musculoskeletal Transplant Foundation, Edison, NJ, USA), placed within the interbody spacer. At 6 and 12 months, radiographic fusion was evaluated as determined by independent radiographic review of angular motion (≤4°) from flexion/extension X-rays combined with presence of bridging bone across the adjacent endplates on thin cut CT scans. In addition other metrics were measured including function as assessed by the Neck Disability Index (NDI), and neck and arm pain as assessed by individual Visual Analog Scales (VAS). The fusion rate for patients using a PEEK interbody spacer in combination with TE was 78.6 % at 6 months and 93.5 % at 12 months. When considering high risk factors, 6-month fusion rates for patients that were current or former smokers, diabetic, overweight or obese/extremely obese were 70 % (7/10), 100 % (1/1), 70 % (7/10), and 82 % (9/11), respectively. At 12 months, the fusion rates were 100 % (12/12), 100 % (2/2), 100 % (11/11) and 85 % (11/13), respectively. Neck function, and neck/arm pain were found to significantly improve at both time points. No serious allograft related adverse events occurred and none of the 31 patients had subsequent additional cervical surgeries. Patients undergoing single-level ACDF with TE in combination with a PEEK interbody spacer and supplemental anterior fixation had a high rate of fusion success without serious allograft-related adverse events.