Objective: To investigate the expression and clinical significance of chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) in hypopharyngeal squamous cell carcinoma (HSCC). Methods: Ninety cases of HSCC patients admitted to our hospital from January 2017 to November 2022 were taken as the study objects, and the HSCC tissues and adjacent tissues of HSCC patients were taken as the experimental materials. Immunohistochemical staining was applied to detect the expression of CXCR4 and CXCL12 in HSCC tissues and adjacent tissues; the levels of CXCR4 mRNA and CXCL12 mRNA in HSCC tissues and adjacent tissues were detected by real-time fluorescence quantitative PCR (qRT-PCR); the clinicopathological characteristics of HSCC patients were collected and recorded, the relationship between CXCR4 and CXCL12 expression levels and the clinicopathological characteristics of patients was analyzed; Pearson was applied to analyze the correlation between CXCR4 and CXCL12 levels. Results: The high expression rate of CXCR4 in HSCC tissues was 60.00%, which was obviously higher than that in adjacent tissues (17.78%) (P<0.05); The high expression rate of CXCL12 in HSCC tissues was 57.78%, which was obviously higher than that in adjacent tissues (24.44%) (P<0.05). The levels of CXCR4 and CXCL12 in HSCC tissue were related to lymph node metastasis, TNM stage and differentiation (P<0.05). Compared with the adjacent tissues, the levels of CXCR4 and CXCL12 in HSCC tissues were obviously higher (P<0.05). Pearson analysis showed that CXCR4 level was positively correlated with CXCL12 level in HSCC patients (r=0.538, P<0.05). Conclusion: CXCR4 and CXCL12 are highly expressed in HSCC tissues, which are related to lymph node metastasis, differentiation degree and TNM stage of HSCC.