Japonica rice ( Oryza sativa , L.), Fangxin 4 was cultured under ambient night temperature (ANT) and high night temperature (HNT) in greenhouse. The average HNTs were 4.7 °C and 8.6 °C greater than ANTs in 2009 and 2010, respectively. The effects of HNT on formation of amyloplast, crystalline structure, thermal properties, and molecular structure were investigated during grain filling stage of 10–35 d after anthesis. Results indicated that amylose contents were gradually formed along with the maturing of rice amyloplast. Moreover, amylose content of HNT treatment decreased by 7.13%–15.44% compared with that of ANT at 35 d after anthesis. T O , T P , T C and Δ H gel for HNT were higher than those of ANT. From 10 to 35 d, relative crystallinity presented an increase–decrease–increase pattern, and the relative crystallinity of HNT was 4.06%–14.51% higher than that of ANT at 35 d after anthesis. Amylopectin under ANT had a higher percentage of degree of polymerization (DP) 6–11, while amylopectin under HNT had a higher percentage of DP 12–23. HNT stress had an influence on the formation of amylose content and amylopectin structure, and then changed the crystalline and thermal properties of rice starch.