To study the expression and spatial localization of Cx43 protein in different differentiated esophageal cancer cells and its effect on the biological behavior of esophageal cancer cells. The expression and distribution of Cx43 protein fluorophores in esophageal cancer cells were detected by image analysis of single particle tracking (SPT)with appropriate fluorescent probe and laser scanning confocal microscope. The distribution of Cx43 in EC9706 cells of highly differentiated esophageal squamous cell carcinoma was similar to that in esophageal carcinoma EC109 cells, especially the cell membrane showed obvious fluorophores. The content of Cx43 protein in the near-cell membrane was higher than that in the cytoplasm, which had the structural basis for building cell-cell communication of the cytomembrane and indicated the trend of benign development of the cell. The fluorescences of Cx43 protein in KY150 cells of poorly differentiated esophageal squamous cell carcinoma and SHEEC cells of esophageal carcinoma were mainly distributed in the cytoplasm near the nuclear membrane, but less distributed near the cell membrane. The content of Cx43 protein in the near-nuclear membrane was higher than that in the near-nuclear membrane, indicating the retention of Cx43 protein in the cytoplasm. Studies have suggested that SPT images can visually trace the distribution of fluorophore of Cx43 protein in esophageal cancer cells, so as to determine the trace of expression and localization of Cx43 protein in different differentiated esophageal cancer cells.