Purpose <p>A significant proportion of patients with head and neck squamous cell carcinoma (HNSCC) are ineligible for immune checkpoint inhibitors (ICIs) because of low programmed cell death protein-ligand 1 (PD-L1) expression. The therapeutic potential of B7-H4 (<i>VTCN1</i>) was investigated using immunohistochemistry (IHC) and spatial transcriptomics (ST).</p> Methods <p>IHC analysis of B7-H4, PD-L1, CD3, CD4, and CD8 was performed using a tissue microarray [94 HNSCC, 94 adjacent squamous intraepithelial neoplasia (SIN), and 69 adjacent normal oral mucosa (NOM) samples]. B7-H4 and PD-L1 expression levels were assessed using tumor cell score (TC; positive, TC &gt; 1%), immune cell score, and combined positive score. ST was performed on six HNSCCs with paired SINs and NOMs to confirm the expression and distribution of B7-H4 (<i>CTVN1</i>), PD-L1 (<i>CD274</i>), CD4 (<i>DC4A</i>), and CD8 (<i>CD8</i>).</p> Results <p>In HNSCCs, TCs revealed a mutually exclusive B7-H4/PD-L1 expression pattern in 55% of samples (<i>p</i> &lt; 0.05). B7-H4 positive TCs were more frequent in HNSCCs (79%) than in SINs (10%) and NOMs (2%). ST analysis confirmed mutually exclusive <i>VTCN1</i>/<i>CD274</i> upregulation in 83% of samples (<i>n</i> = 6) and demonstrated co-localization of B7-H4 protein and <i>VTCN1</i> in IHC-positive areas. B7-H4 was significantly correlated with low-CD8⁺ T-cell infiltration (<i>n</i> = 94, <i>p</i> = 0.009), and <i>CD8A</i> mRNA was down-regulated in the <i>VTCN1</i>⁺ area compared with that in the <i>VTCN1</i>⁺ area.</p> Conclusion <p>B7-H4 is a promising antibody–drug conjugate target in ICI-resistant HNSCC. IHC combined with TCs enabled the reliable assessment of B7-H4, given its co-localization with <i>VTCN1</i> in IHC-positive areas and association with low-CD8⁺ T-cells.</p>