The transient transformation system plays an important role for initially and rapidly verifying gene functions and identifying related phenotypes. To improve the transformation efficiency and stability of transient transformation system of the Chinese chestnut callus and to identify the function of the Chinese chestnut starch synthase gene (CmSSⅠ), we optimized the transient transformation system of Chinese chestnut callus through different states of callus tissues and expression vectors by using the callus tissues induced from young embryos of Chinese chestnut ‘Yanshanhongli’. The results showed that: (1) callus was divided into an embryogenic callus (EC_E) and early stage of embryogenic callus (EC_D Ⅰ, Ⅱ and Ⅲ) based on the difficulty level of initiating somatic embryo. The early stage of embryogenic callus with white and relatively dispersed texture (EC_D Ⅲ) were more suitable for the transient transformation system of Chinese chestnut callus. (2) The transient transformation efficiency of RNAi silencing vector pK7GWIWG2 (Ⅱ) RR277 was the highest at 87.67% when the transformed material was at the early stage of embryonic callus (EC_D Ⅲ). (3) The expression of CmSSⅠ gene was significantly downregulated in the transgenic callus tissues by the optimal callus transient transformation system, and the contents of total starch and amylopectin of the CmSSⅠsilenced positive callus were significantly reduced, and amylose content was not significant differences. In this study, the efficiency and stability of the transient transformation system of Chinese chestnut callus tissues were improved, and it was demonstrated that the gene CmSSⅠ was regulating the synthesis of Chinese chestnut starch. This laid a foundation for the study of Chinese chestnut gene function and further provided a technical platform for molecular assisted breeding.