Resistant starch (RS) from uncooked and cooked high amylose starch (HAS) was used to investigate the effect of RS structure on the production of short-chain fatty acids (SCFA) using a static anaerobic in vitro system. The pH value of culture correlated well to the production of total SCFA after the fermentation (R2 = 0.969). Most importantly, fermentation of RS from thermally treated starch under no moisture condition produced the highest concentration of SCFA and the greatest ratios of butyrate/acetate and butyrate/total SCFA, followed by the fermentation of RS from uncooked HAS. FTIR analysis suggests that all the RS exhibited a relatively higher organized structure compared to its corresponding original starch. The analysis of molecular structure by HPLC showed that the most pronounced difference in molecular composition of RS between the group favoring greater butyrate production and the group supporting a lower butyrate production was that larger molecules (amylopectin fraction) still existed in the former group but not in the latter group. Thus, it can be concluded that RS structure, in particular molecular structure is one of the key factors manipulating SCFA production in amount and proportion.