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Objective: To investigate the value of combined assay of serum pepsinogen (PG) and osteopontin (OPN) levels for gastric cancer screening. Methods: Fasting serum pepsinogen Ⅰ, Ⅱ and OPN concentration were measured by ELISA in 570 subjects. The cut off points for PG and OPN were determined using ROC. Results: Using serum PGI concentration ≤80 ng/ml, Ⅰ:Ⅱ ration ≤5.0 and OPN concentration ≥34 ng/ml or ≥30.44 ng/ml (based on ROC)for gastric cancer screening had superior specificity, positive and negative predictive values compared with PG or OPN screening only. The sensitive and specificity of combining serum PG and OPN assay were both more than 70% compared with PG or OPN screening alone. Conclusion: Combining serum PG and OPN assay for gastric cancer screening is superior to PG or OPN level only. This may be a new method for gastric cancer mass screening.