Background: The primary treatment for hepatocellular carcinoma (HCC) is radical surgery. However, HCC has a high propensity to recur postoperatively and no standard adjuvant therapies have been approved. This Phase I trial aims to evaluate the safety and efficacy of an adjuvant personalized neoantigen pulsed autologous dendritic cell vaccine (Neo-DCVac-02) in patients with HCC. Methods: Eligible patients had histologically confirmed high-risk HCC after radical surgery. After obtaining the necessary baseline biopsy specimens and PBMCs of appropriate quality for DNA and RNA sequencing, neoantigen prediction, screening and synthesis were performed. Subsequently, leukapheresis was performed and DCs were isolated and cultured. DCs were then pulsed with neoantigen peptides to produce Neo-DCVac-02. On day 0, patients were treated with cyclophosphamide at a dose of 250 mg/m2. The prepared Neo-DCVac-02 vaccine was administered subcutaneously to the axillary and inguinal regions bilaterally on day 1, followed by daily administration of GM-CSF at a dose of 0.075 mg for 5 days (days 2-6). The primary objectives of this study were to assess feasibility and safety/tolerability. Secondary objectives included evaluation of immune responses (via vaccine response: IFNγ ELISpot), relapse-free survival (RFS) and overall survival (OS). Results: A total of 32 patients provided informed consent to initiate the process of personalized neoantigen discovery, of which 26 (81%) met the requirements for successful product selection for clinical manufacturing. Feasibility was demonstrated with 13 patients receiving Neo-DCVac-02. 13 patients did not receive the neoantigen vaccine (7 due to progressive disease, 6 due to withdrawal of informed consent because of COVID-19). The treatment was well tolerated with no grade 2+ treatment-related adverse events (TRAEs). The most common Grade 1 TRAEs were injection site reactions (75%), rash (16.7%), fatigue (8.3%), neutropenia (8.3%), and headache (8.3%). Five of 13 evaluable patients showed increased antigen-specific T-cell activity and more than 50% of the neoantigen peptides in Neo-DCVac-02, and sustained cellular responses were observed up to one year. Increases in T-cell activation/co-stimulation seen after treatment with Neo-DCVac-02, as demonstrated by flow cytometric analysis, suggest immune priming. At a median follow-up of 29.7 months, median RFS was not reached, with 1- and 2-year RFS of 84.6% and 60%, respectively, and all patients are alive. Patients with a strong immune response had a longer median relapse-free survival (not reached) compared to patients with a weak immune response (17.6 months, P = 0.003). Conclusions: These data demonstrate that Neo-DCVac-02 is safe, well tolerated and capable of inducing durable antigen-specific lymphocyte immune responses that may correlate with delayed HCC recurrence. Clinical trial information: NCT04147078.