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To date, few research have been reported on the evolution of C-3/C-4 vegetation in southern China, and the main mechanism influencing the evolution of C-3/C-4 vegetation is unclear. That makes it difficult for researchers to understand the competition mechanism of C-3 and C-4 plants under different climate environments and its relationship with the climate factors. It is also not conducive for researchers to assess the influence of future climate change on regional C-3/C-4 vegetation. Exactly, C-3/C-4 vegetation change in the regional-scale will have a significant impact on the global carbon cycle and agricultural production. Therefore, it is especially important to reconstruct the evolutionary history of C-3/C-4 vegetation in southern China and clarify the influencing mechanism of climate change. In this study, we systematically analyzed stable carbon isotope (delta C-13) of the long-chain n-alkanes in sediment samples from eight lakes and four peat profiles in southern China, to reconstruct the spatiotemporal evolution of C-3/C-4 vegetation in these regions since the Last Glacial Maximum (LGM) and to investigate the climate factors that affect the C-4 plant abundance change in the research area. The integrated long-chain n-alkane (C-27-C-33) stable carbon isotope results of samples from Zhanjiang Huguangyan Maar Lake, Xingyun Lake, Lugu Lake and Dingnan peat showed that, from the LGM to the Early Holocene, C-4 plant relative abundance exhibited a gradually increasing trend from 21% to 34%. In the Middle Holocene, the C-4 plant abundance significantly declined and reached a lowest value of 10%. In space, the C-4 plant abundance generally exhibited a gradually declining trend from south to north in the LGM and the Early Holocene, while it showed an opposite trend in the Holocene Climate Optimum (6.0 cal ka BP). It reflected that the main influencing factor on C-4 plants spatial distribution was changing from temperature to precipitation. This study indicated that temperature was the dominant factor affecting C-4 plant distribution in southern China, however, when the temperature condition was appropriate, an increase in precipitation (especially more spring precipitation) would reduce the competitive advantages of C-4 plants. Therefore, the combination of temperature and seasonal precipitation was the important factor that determines the C-3/C-4 vegetation proportion change in the southern China. Under the premise that the temperature will rise and precipitation will increase in the future, the reduction of a competitive advantage for the C-4 plants could affect agricultural production in China.