The Yangjiang-Yitongdong Fault (YJF) is an important NW-trending regional fault, which divides the Zhujiang (Pearl) River Mouth Basin (ZRMB) into western and eastern segments. In Cenozoic, the northern continental margin of the South China Sea (SCS) underwent continental rifting, breakup, seafloor spreading and thermal subsidence processes, and the Cenozoic activities of YJF is one part of this series of complex processes. Two long NW-trending multichannel seismic profiles located on both sides of the YJF extending from the continental shelf to Continent-Ocean Boundary (COB) were used to study the tectonic and sedimentary characteristics of western ZRMB. Using the 2D-Move software and back-stripping method, we constructed the balance cross-section model and calculated the fault activity rate. Through the comprehensive consideration of tectonic position, tectonic evolution history, featured structure, and stress analysis, we deduced the activity history of the YJF in Cenozoic. The results showed that the YJF can be divided into two segments by the central uplift belt. From 65 Ma to 32 Ma, the YJF was in sinistral motion as a whole, inherited the preexisting sinistral motion of Mesozoic YJF, in which, the southern part of YJF was mainly in extension activity, controlling the formation and evolution of Yunkai Low Uplift, coupled with slight sinistral motion. From 32 Ma to 23.8 Ma, the sinistral motion in northern part of YJF continued, while the sinistral motion in southern part began to stop or shifted to a slightly dextral motion. After 23.8 Ma, the dextral motion in southern part of YJF continued, while the sinistral motion in northern part of YJF gradually stopped, or shifted to the slightly dextral motion. The shift of the YJF strike-slip direction may be related to the magmatic underplating in continent-ocean transition, southeastern ZRMB. According to the analysis of tectonic activity intensity and rift sedimentary structure, the activities of YJF in Cenozoic played a regulating role in the rift extension process of ZRMB.