Abstract: Objective To observe the influence of pretreatment solution temperature on the ultrastructure of myocardiocytes in sample preparation of the transmission electron microscope(TEM), so as to determine the optimal experimental temperature. Methods A total of 15 healthy C57/BL6 mice were randomly divided into three groups (n=5 each). Under the intraperitoneal anesthesia, myocardial tissue was obtained from each mouse and the routine sample preparation procedure of TEM was carried out. Processes of rinsing, post-fixing and dehydrating were carried on at 4℃ in group Ⅰ, 20℃ in group Ⅱ and 37℃ in group Ⅲ. Results TEM results showed that the ultrastructure of myofibrils, mitochondria, submucosal vesicles and Z lines in group Ⅰ were well preserved and the sample background was clear and neat; in group Ⅱ, myofibrils were neat, a few membranous structures of mitochondria and submucosal vesicles were dissolved, a few black particles were attached to Z lines, and the interstitial space was contaminated by black particles; in group Ⅲ, the structure was poorly preserved, myofilament was blurred, part of the mitochondria and most of myolemma vesicles membrane were dissolved, submucosal vesicle membrane structure was dissolved and was not clear, and black particles were adhered to Z lines and the interstitial space was heavily polluted by black particles. Conclusions The preservation of the ultrastructure of myocardiocytes during sample preparation of TEM was influenced by the temperature of the pretreatment solution, and the most optimal experimental temperature is 4℃.