Rapeseed–rice rotation is a cropping system that improves the land-use rate. The present study conducted a field experiment with winter planting of rapeseed as treatment (WR) and winter fallow as control (CK) to investigate the effects of winter planting of rapeseed on growth, yield formation, and 2-acetyl-1-pyrroline (2-AP) biosynthesis of fragrant rice in the two subsequent cropping seasons. The results show that WR treatment improved alkali-hydro nitrogen and rapidly available phosphorus contents in soil. Compared with CK, WR treatment significantly increased grain yield and effective panicle number per plant by 21.16–27.26% and 7.33–21.24%, respectively. Higher net photosynthetic rate, leaf area index, and dry matter accumulation of fragrant rice plants were recorded in WR treatment and CK. Furthermore, compared with CK, WR treatment significantly increased grain 2-AP content, which could be explained by increased content of pyrroline-5-carboxylic acid, methylglyoxal, 1-pyrroline, and enhanced activity of proline dehydrogenase. In addition, future studies should be conducted at a molecular level to reveal the regulation mechanism in 2-AP biosynthesis of fragrant rice under conditions of winter planting of rapeseed.