Objective To investigate the relationship between claudin-1 and micro-lymphatic vessel density (MLVD) by detecting expressions of claudin-1 and protein D2-40 in hypopharyngeal squamous cell carcinoma (HSCC), and to study the mechanism of lymph node metastasis in HSCC by exploring the correlation between their expressions and clinical stage, pathological grade, and clinical prognosis of HSCC. Methods Expression levels of claudin-1 and protein D2-40 were detected by immunohistochemistry in cancer specimens from 97 patients with HSCC and 90 para-tumor specimens. The correlation between the markers and clinicopathological features was analyzed by using the Pearson chi-square test, and survival analysis by the log-rank test. Results Claudin-1 expression was high in HSCC and was related to tumor differentiation (P=0.004) and lymph node metastasis (P=0.026). Kaplan-Meier analysis showed that claudin-1 expression was related to patients' survival rate (P=0.003). There were significant relationships between MLVDs in the para-tumor tissues and the indices of histopathological grade, clinical stage, and lymph node metastasis. The claudin-1 expression was positively correlated to MLVD (r=0.54, P=0.012). Conclusion High expression of claudin-1 may induce the generation of tumor lymphatic vessels and facilitate lymphatic metastasis in HSCC, and therefore becomes a new target for the treatment of this tumor.