Abstract: Objective To observe the effects of temporary acid exposure on cell proliferation and extracellular signal-regulated protein kinase (ERK) activity in normal human esophageal epithelial cells in vitro. Methods Normal human esophageal epithelial cells cultured in vitro were exposed to acidic media (pH 4.0-6.5) for 3 to 60 min, and the control cells were cultured at pH 7.3. MTT assay and flow cytometry were employed for cell proliferation assessment. The expression of phosphorylated ERK1/2 protein was determined by immunoblotting. Results Esophageal epithelial cells with acid exposure for 3 min exhibited a significant increase in cell proliferation, increased number of cells in S phase and enhanced expression of phosphorylated ERK1/2 protein. Acid exposure of the esophageal epithelial cells exceeding 6 min resulted in depressed proliferation and decreased S-phase cells, and cell proliferation induced by acid exposure was abolished by pretreatment with U0126. Conclusion Temporary acid stimulus increases cell proliferation of normal human esophageal epithelial cells in vitro by activating the ERK pathway.