Abstract: Objective To investigate the distribution of myosin, heavy polypeptide 9(MYH9) protein in the mitochondria of esophageal squamous cell carcinoma(ESCC), and the interaction between MYH9 protein and a mitochondria-associated protein, stomatin-like protein 2(SLP-2). Methods In order to find the binding protein of a new reported mitochondria-associated protein SLP-2, co-immunoprecipitation(Co-IP) was conducted by anti-SLP-2 antibody in the lysate of ESCC cell lines KYSE150. Precipitated mixture was analyzed by matrixassisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Co-IP and glutathione S transferase(GST) pull down assay were performed to further confirm the result of MALDI-TOF-MS. To detect the distribution of MYH9 in ESCC mitochondria, sucrose density gradient centrifugation was adopted to isolate the mitochondria for Western blot. Results In the lysate of ESCC cell lines KYSE150，MYH9 protein coimmunoprecipitated with SLP-2 protein and binded with GST-SLP-2. MYH9 protein existed in both isolated cytoplasm and purified mitochondria of ESCC cell lines KYSE150. Conclusion Parts of MYH9 distributes in the mitochondria of ESCC and bind with a mitochondria-associated protein SLP-2. MYH9 may affect the function of ESCC mitochondria.