The middle reaches of the Huai River was a key zone for the exchange, collision, and fusion of ancient cultures between the Yangtze and Yellow River basins and between the eastern coast and western hinterland. Between 5.0 and 4.3 ka BP, the Late Dawenkou culture from the Haidai region moved south, which brought the Neolithic culture of this area into a new stage of development and initiated the agricultural transformation from mono-rice cultivation to mixed rice-and-millet cultivation. However, the characteristics of the Late Dawenkou culture’s agroeconomy and its influencing factors are unclear. Thus, this study used the Gaixia site in Guzhen County, Anhui Province, as the object of study and performed analysis of charred plant remains and AMS 14C dating. Results showed that a mixed pattern centered around rice cultivation emerged during the Late Dawenkou period. Further, the analysis of rice grain shape and spikelet bases indicated the rice remains correspond to the small-grained japonica rice variety with a high degree of domestication; this rice type may have been preferred because of its uniform harvest time or greater environmental adaptability. Combined with existing archaeobotanical, paleoenvironmental, and cultural analyses, we can conclude that overall, the Late Dawenkou culture in the middle reaches of the Huai River had an agricultural economy dominated by rice and supplemented by millet. Environmental changes, agricultural traditions, and cultural exchange jointly influenced this agricultural structure. Finally, geomorphological factors may be behind differences in the relative proportions of rice and millet cultivation at different sites in the region.