Abstract: Objective To investigate the involvement of gap junction protein 43 (CX43) and tumor necrosis factor (TNF) - αin mice with acute visceral pain in spinal dorsal horn astrocytes. Methods Healthy male C57BL/6 mice were randomly divided into an acetic acid (AA) group and a normal saline (NS) group. A mouse model of acute visceral pain was established through intraperitoneal injection of 0.6% acetic acid in the AA group, while normal saline was injected into the NS group. To further explore the involvement of CX43 and TNF-α in acute visceral pain, each group was divided into following subgroups: intrathecal injection of antagonists (AA + CBX and NS + CBX; AA + anti-TNF-αand NS + anti-TNF-α) and NS (AA + NS and NS + NS). The behavioral changes of mice were observed by measuring the time of twisting. The expression of connexin 43 (CX43) was detected by Western blot. The activation of astrocytes in the spinal dorsal horn was detected by immunofluorescence. The expression of TNF-α in the spinal cord was detected by ELISA. Results Compared with the NS group, the AA group presented activated astrocytes and remarkably increased levels of TNF-α (P<0.01), without significantly exchanges in the expression of CX43 protein (P>0.05). After intrathecal microinjection of CBX or anti-TNF-α, the activation of astrocytes was inhibited, the level of TNF-α and the time of twisting significantly reduced (P<0.01) and acute visceral pain in mice was relieved. Conclusions CX43 regulates the release of TNF-α in spinal dorsal horn astrocytes of mice, so as to invovle in acute visceral pain induced by intraperitoneal injection of acetic acid.