Seed development is one of the most important agricultural traits, determining both the crop yield and quality of fleshy fruits. A typically abortive litchi cultivar, Guiwei, exhibits heterogeneity in seed size across production areas, years, and individual trees. Previous studies have shown that 'Guiwei' seed development failure is associated with endosperm arrest and chilling conditions. Herein, we identified a cysteine proteinase inhibitor (LcCPI5) and defensin-like protein (LcDEFL) as key genes in determining 'Guiwei' seed development through combined analysis of 'Guiwei' and 'Huaizhi' endosperm single-nucleus RNA-sequence and transcriptome data of 'Guiwei' seed subjected to different temperature treatments. LcCPI5 was exclusively expressed in the endosperm sample of 'Guiwei' at 25 days post-anthesis, and its expression decreased in response to chilling. The silencing of LcCPI5 led to significantly larger seeds, whereas employing the cysteine proteinase inhibitor E-64 resulted in smaller seeds in the cultivar 'Guiwei'. Unlike LcCPI5, LcDEFL promotes litchi seed development. The large seed cultivar 'Huaizhi' had substantially higher expression of LcDEFL than the partly abortive cultivar 'Guiwei'. LcDEFL silencing led to a notable reduction in the size of litchi seeds. These findings point to the post-translational modulation of cysteine proteinase and the critical role of cysteine-rich proteins in litchi seed development.