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To elucidate the role of substitution of arabinoxylan (AX) in the characteristics of wheat starch, this study prepared AX with a well-defined structure by targeted enzymatic hydrolysis and comparatively investigated the effects of AX with different degrees of substitution on gelatinization and retrogradation behavior of starch. Removal of major arabinofuranosyl (Araf) of monoor disubstituted xylopyranosyl (Xylp) of both low-molecular-weight (Mw: 62.5 kDa, Araf/Xylp: 0.61) and high-molecular-weight AX (Mw: 401.2 kDa, Araf/Xylp: 0.61) reversed the decreased gelatinization viscosity and recrystallization of amylose induced by AX to a similar extent. Upon retrogradation for 30 days, the Araf of mono-and disubstituted Xylp contributed to the water distribution and the effect depended on the molecular chain length. The C3-linked Araf of disubstituted Xylp was more involved in prohibiting the retardation of recrystallization of amylopectin, while the presence of Araf of monosubstituted Xylp might hinder the interactions between AX and amylopectin.