Fault geometry, kinematics, geophysics, the tectonic stress field and tectonic evolution of the Zhouwang fault in the southern Jiangnan tectonic transition zone of the Lower Yangtze region, eastern China are examined. Field observations show the fault is composed of a series of nearly E–W trending, N–S dipping faults, and four stages of tectonism (sinistral strike-slip, thrust nappe, normal fault, and dextral strike-slip) developed in turn. Geophysical data show that the fault trends almost linearly E–W along a flat, steep gravity gradient at shallow depth, with distinct gravity anomalies to the north and south and different in the north and south. Also, the deep part is characterized by northward dip and a gradual slowing down. Tectonic stress field analysis indicates that the fault experienced four tectonic movements: NNE–SSW compression, NNW–SSE compression, NEE–SWW extension, and E–W compression. Combined with regional tectonic background and previous research results, this indicates that: (1) the Zhouwang fault experienced sinistral strike-slip movement during the Indosinian Period (260–200 Ma); (2) thrust nappes developed during the early Yanshanian Period (163–145 Ma); (3) a normal fault occurred in the late Yanshanian Period (125–65 Ma); and (4) dextral strike-slip movement occurred in the Himalayan Period (ca. 50–37 Ma). The results reveal the tectonic evolution of the fault during Mesozoic deformation in the area, and also reveal the geological evolution and tectonic transformation of the Lower Yangtze region, which is key to our understanding of intracontinental deformation in eastern China.