Abstract: Objective：To establish a monoclone cell line of squamous cell carcinoma（SCC）in rat tongue mucosa and to study its biological characteristics. Methods：SCC in rat oral mucosa was induced by adding 4-nitroquinoline-1-oxide (4NQO) to the SD rat’s drinking water, and the cancer tissue was then cultured to obtain mixed cells in vitro. Mono cell cloning method was used for purification of the mixed tumor cells. The biological characteristics of the cells were studied by microscope observation, CCK-8 Cell Counting Kit, plate clone formation assay, flow cytometry assay and immunohistochemistry staining. Hypodermic inoculations of the cells in nude mice were performed to observe the tumor formation ability. Results：The tumor cells were spindle-shaped and expressed the proteins of cytokeratin, Vimentin and N-cadherin. The cellular population doubling time was 23.35 hours. The colony forming efficiency was 63.06% and the xenograft rate was 100％ in nu/nu BALB/C mice. Conclusions：Rca-T, an oral squamous cell carcinoma cell line derived from Sprague-Dawley rat tongue mucosa carcinoma, was established successfully. This cell line would be a model for basic and clinical research of oral cancer.