The present study aimed to investigate whether procaine may upregulate the expression of aquaporin-5 (AQP5) in human salivary gland ductal cells and the underlying mechanisms of this upregulation. Immortalized normal human salivary gland ductal cells (NS-SV-DC), lacking AQP5 protein expression, were used to measure the glandular secretion rate following treatment with procaine, and the protein expression levels of AQP5 in NS-SV-DC cells were measured by western blotting. In order to investigate the mechanism of procaine action on AQP5 protein expression, the protein expression and activity of DNA methyltransferase (DNMT)1, and the CpG methylation of AQP5, were investigated further. In NS-SV-DC cells treated with procaine, the mRNA and protein levels of AQP5, and the secretion rate of cells, were significantly increased. Although no significant alterations were observed in the protein expression of DNMT1 following procaine treatment, its enzymatic activity was reduced, resulting in CpG island demethylation at Sp1-2 and Sp1-3 sites of the AQP5 gene, which may contribute to the significantly upregulated AQP5 gene expression. The results of the present study indicate that procaine may upregulate the protein expression of AQP5 in human submandibular glands by inhibiting the activity of DNMT1 and promoting liquid secretion. The procaine-mediated expression of AQP5 may provide a novel regimen for the treatment of SS syndrome.