Background <p>Proliferating cell nuclear antigen (PCNA), a well-documented anticancer target, is critical for DNA synthesis, replication, and repair. AOH1996, a small-molecule PCNA inhibitor, is currently undergoing clinical trials for the treatment of advanced solid tumors. However, the therapeutic effect of AOH1996 on head and neck squamous cell carcinoma (HNSCC) remains unclear.</p> Methods <p>The effects of AOH1996 on HNSCC biological behaviors and cancer stemness were tested in HNSCC cells and nude mice. The combination treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing, Western Blotting, immunofluorescence staining, comet assays, and qRT‒PCR were conducted for mechanistic studies.</p> Results <p>Our results showed that AOH1996 effectively inhibited HNSCC proliferation and invasion both in vitro and in vivo. AOH1996 suppressed HNSCC stemness, development, and metastasis. Moreover, AOH1996 altered the tumor immune microenvironment into an inflamed state with increased CD8⁺ T-cell infiltration, rendering it a favorable partner for combination therapy with immune checkpoint inhibitors. Mechanistically, AOH1996 induced cellular DNA damage, suppressed cancer stemness through the upregulation of p-TBK1, and promoted the secretion of CD8⁺ T-cell-recruiting chemokines by stimulating IRF3-mediated transcription.</p> Conclusions <p>Taken together, our results demonstrated that AOH1996 suppressed tumor growth, eliminated cancer stem cells (CSCs), and synergistically enhanced the efficacy of anti-PD1 immunotherapy in HNSCC.</p>