We evaluated the efficiency of transfecting adenoviral vectors encoding enhanced green fluorescent protein (AdCMV-EGFP) into rat submandibular gland cells and the effects of gene transfer on cell proliferation and secretory function. Isolated submandibular gland cells were transfected with different titers (or multiplicity of infection, MOI) of AdCMV-EGFP. The transfection efficiency was evaluated by quantifying EGFP-positive cells by inverted fluorescence microscopy, cell proliferation by MTT assay, and cell secretory activity by measuring α-amylase in culture medium. A transfection efficiency of up to 70.8% was achieved in submandibular gland cells. MTT assay showed that increased viral titers resulted in significant inhibition of cell proliferation, which occurs on day 5 post-transfection. Simultaneously, the amylase levels started to reduce with a significant decrease on day 7 after transfection. The results show that AdCMV-EGFP transfection of submandibular gland cells at higher MOI results in cytotoxicity, decreased cell proliferation, and secretory function. However, the lower adenoviral titers (e.g., 200 particles/cell) could be an efficient and safe labeling tool for gene transfer to submandibular gland cells.