To investigate the fine starch structure characteristics and formation mechanism of high-quality appearance soft rice, two high-quality and low-quality soft rice varieties (HA-SR and LA-SR, respectively) were selected. Differences in appearance quality, fine starch structure, and activity of key enzymes involved in starch synthesis during the grain-filling stage were compared. The results showed that compared with LA-SR, HA-SR were less chalky, more transparent, had larger starch grains, a lower content of shorter chains (DP 6-24), a higher content of longer chains (DP ≥ 25), lower relative crystallinity, fewer ordered structures, more amorphous structures and larger thicknesses of semi-crystalline lamellae. In terms of amylase activity during the grain-filling stage, the AGPase and GBSS activities of HA-SR were higher, and the SBE activity of HA-SR was lower compared to LA-SR. In conclusion, higher AGPase activity can produce a higher filling rate resulting in fuller starch grain in soft rice. Fuller starch grains reduce the chalkiness of soft rice. Higher AGPase and GBSS activities and lower SBE activity can result in soft rice with more long-branched and less short-branched amylopectin. Thus, soft rice has lower relative crystallinity and less ordered structure. These structures may facilitate reduce grain chalkiness and improve grain transparency.