AIM: To investigate the pathological features of ocular surface squamous neoplasia (OSSN) and evaluate the synergistic therapeutic effects of interferon-α2b (IFNα2b) and 5-fluorouracil (5-FU) on cellular proliferation, migration, apoptosis, and cell cycle of human oral squamous carcinoma cell line Cal27. METHODS: Tissue specimens from OSSN were processed with hematoxylin-eosin (HE) and immunofluorescence (IF) staining to characterize pathological changes. We analyzed the expression levels of four pivotal proteins involved in 5-FU metabolism: interferon alpha receptor (IFNAR), thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD). Cal27 cell lines were treated with a spectrum of concentrations of IFNα2b and 5-FU, either in isolation or in combination. Then, cell activity was measured utilizing CCK-8 assay and dose-effect curves were calculated, while tumor cell migration was detected by cell scratch experiments. Cal27 cells were added with IFNα2b and 5-FU in a non-constant ratio drug combination design and the corresponding combination index (CI) and fraction affected (Fa) were calculated with CompuSyn software. Western blot assay was conducted to quantify the expression of TP, TS, and DPD. Cell cycle and apoptosis were measured with flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. RESULTS: Treatment with both IFNα2b and 5-FU inhibited cell proliferation. Except for the lowest and highest doses of 5-FU, CI values for all other groups were below 1, suggesting a synergistic interaction. Low concentrations of IFNα2b and 5-FU both diminished the relative mobility of Cal27 cells, instead, a stronger inhibitory effect was observed when the two drugs were co-applied. The expression levels of TP and DPD in Cal27 cells were dose-dependently increased at a low concentration of IFNα2b. Low-dose IFNα2b combined with 5-FU significantly inhibited cell proliferation in G0/G1 phase compared to 5-FU monotherapy. Medium and high doses of IFNα2b and all concentrations of 5-FU could induce apoptosis in a concentration-dependent manner. The susceptibility to 5-FU treatment and apoptosis rates of tumor cells were elevated with low doses of IFNα2b. CONCLUSION: Both IFNα2b and 5-FU, when administered individually or in combination, effectively suppress the proliferation and migration of Cal27 tumor cells, induce cell apoptosis and arrest cell cycle. Low doses of IFNα2b increase the antitumor effects of 5-FU on Cal27 potentially through up-regulating the expression of TP, demonstrating a synergistic effect between IFNα2b and 5-FU.