Cultivated land fragmentation (CLF) is a key obstacle to agricultural development and has a strong relationship with regional food security and global sustainable development. However, few studies have analyzed the spatio-temporal distribution pattern and evolution characteristics of CLF and the complex interactions among their influencing factors in rapidly developing regions. In this study, first, the GlobeLand30 datasets were used to obtain characteristic parameters of cultivated land in counties in Guangdong Province in 2000, 2010, and 2020. Then, the linear weighted comprehensive evaluation model based on the principal component analysis (PCA) was used to measure the extent of CLF. Finally, the exploratory spatial data analysis (ESDA) was used to analyze the spatio-temporal distribution pattern and evolution characteristics of CLF, and geodetector (GD) and random forest (RF) models were used to explore the factors influencing the spatial difference in CLF. The results showed that the spatial differences in the distribution of cultivated land resources in Guangdong Province are relatively large and the extent of agglomeration is generally low. The extent of CLF on the county scale is mainly medium and higher. The overall spatial distribution shows an increasing trend from the south to the north and from the west to the east, and the spatial distribution pattern with agglomeration and randomness remains relatively stable. From 2000 to 2020, the overall CLF continued to intensify and the evolution of CLF on the county scale mainly increased. The spatial difference in CLF is the result of that based on the natural environment and influenced by factors such as social, economic, and agricultural development. The interaction between influencing factors is very strong, dominated by nonlinear enhancement. The results are of great significance for promoting the intensive and efficient utilization of cultivated land resources and sustainable regional development.