Esophageal carcinoma (EC) is a common malignancy and the major determinant of overall cancer mortality in A-sia, with bone as one of the most frequent metastasic sites. The aim of this study was to establish a Chinese esophageal squamous cell carcinoma (ESCC) cell line CEK-Sq-1 with high bone metastasis potency usingTc-MDP micro pinhole bone scintigraphy and micro PET/CT for exploring the mechanism of occurrence and development in EC.The cell line came from the surgical tissue of a sixty-one year-old male Chinese patient with ESCC. BALB/c nu/ nu immunodeficient mice tumorigenicity was confirmed. The cell growth curve was mapped and analysis of chromosome karyotype was done. Approximately 1×106 cancerous cells were injected into the left cardiac ventricle of immunodeficient mice. The bone metastatic lesions of tumor-bearing mice were detected by Tc-MDP bone scintigraphy with a self-designed micro pinhole and were resected from the mice under deep anesthesia. The bone metastatic cells in the lesions were used for culture and then for repeated intracardiac inoculation. This in vivo-in vitro-in vivo experimental metastasis study was repeated for four cycles to obtain a novel subline, CEK-Sq-IBM. All of the suspicious bone sites were confirmed by pathology. Real-time polymerase chain reaction (PCR) was used to compare the gene expression in the parental cells (CEK-Sq-1) and in the bone-seeking cell subline (CEK-Sq-IBM).First passage cells of CEK-Sq-1 were planted in immunodeficient mice subcutaneously and the tumorigenesis rate was 100％. The chromosome karyotype analysis of the cell line was hypotriploid. The bone metastasis rate went from 20% with the first passage cells to 90% after four additional cycles. The established bone-seeking subline named CEK-Sq-IBM had a high potential to metastasize in bone, including mandible, humerus, thoracic vertebra, lumbar, scapula and femur. The bone metastasis lesions were successfully detected by micro pinhole bone scintigraphy, micro PET/CT, and partly by X-ray. A gene expression difference was found between parental and bone-seeking cells.CEK-Sq-1 is a novel Chinese esophageal squamous cell carcinoma cell line which has produced a subline with high bone metastasis potency in nude mice, thereby serving as a useful tool for studying ESCC bone metastasis.