In this study, high-amylose maize starch (HAMS) and four fatty acids with different saturation degrees were used as the raw materials to form starch-unsaturated-fatty-acid complexes. The effects of the different complexes on the in vitro large intestine fermentation characteristics and regulation of microbiota were investigated. The results showed that with the increase of the unsaturation of fatty acids, the formation of V-type starch complexes (30.42%~32.93%) decreased, except for the starch-oleic-acid complex (34.87%). There were no significant differences in the fermentation rate for the starch-unsaturated fat complexes during the entire fermentation process (p>0.05), with their final gas productions being essentially the same (13.8~14.2 mL). The starch-unsaturated fatty acid complexes produced a lower concentration of butyrate (14.83~17.91 mmol/L; compared with HAMS 22.42 mmol/L), but produced higher concentrations of acetate (60.25~63.73 mmol/L) and propionate (21.22~24.81 mmol/L). The starch-unsaturated fatty acid complexes all significantly increased the relative abundance of Prevotella, which might be associated with their higher propionate productions. Taken together, the in vitro large intestine fermentation characteristics of HAMS-unsaturated-fatty-acid complexes were less affected by the fatty acid saturation, but related mainly to their own complex structure. The saturation of the fatty acids in the HAMS-unsaturated fatty acid complexes has a certain effect on the microbiota structure.