As the non-digestible portion of starch, resistant starch (RS) has been associated with many health beneficial effects such as modulating glycemic response and improving gut health. Among several types of RS, a novel type, i.e., RS5, has attracted more attention in recent years due to its high thermal stability and added functionality. The main objective of this study is to develop a V-type starch inclusion complex with retarded digestibility and low glycemic index (GI) as a novel RS5 and elucidate the structure-digestibility relationship in starch inclusion complex formed using different preparation methods and hydrothermal treatments. The starch inclusion complex of interest in this study is that formed with a bioactive guest compound, ascorbyl palmitate (AP). First, AP was shown to significantly reduce the in vitro digestibility of raw, cooked, and retrograded high amylose maize and potato starch. Then, its inclusion complexes with starch were prepared using two methods, i.e., the dimethyl sulfoxide (DMSO) method and the novel “empty” V-type method, and several hydrothermal treatments were applied. Although minimal RS content was present in the raw inclusion complex samples prepared by both methods, hydrothermal treatments significantly increased the RS content. Among all, annealing followed by strong acid hydrolysis (ANN-ACH) was the most effective treatment in enhancing RS content. Lastly, the ANN-ACH starch-AP inclusion complex was further examined for its hydrolysis pattern and GI in both in vitro and in vivo experiments. Consistent results were obtained that this inclusion complex showed the least glucose release and the lowest GI value, falling within the range of low GI foods. The in vivo GI was even lower than that of the raw high amylose maize starch (RS2), indicating its more pronounced effect in modulating the postprandial glycemic response in mice. In combination of the high RS content, moderate hydrolysis pattern, low GI value, and the distinctive V-type crystalline structure, the ANN-ACH treated starch-AP inclusion complex could provide potential beneficial effects on health. Future research is warranted to explore the potential ways of incorporating such ingredient in food products and the long-term effect of such novel RS5 for the prevention and treatment of chronic diseases.