The chemical properties of β-glucans leading to their inhibition on aflatoxin (AF) production by Aspergillus flavus remain unclear. In this study, structurally modified lentinan derivatives were prepared by carboxymethylation, sulfation, and phosphorylation to explore their inhibition activity to AF synthesis. The results demonstrated that inhibitory activity of lentinan decreased at higher or lower concentrations than 200 μg/mL. Compared with lentinan, the sulphated derivatives only performed a reduced optimal inhibition rate at a higher concentration. The phosphorylated derivatives achieved complete inhibition of AF production at 50 μg/mL, but the inhibitory activity was attenuated with an increase of concentration. The minimum concentration of carboxymethylated derivatives to completely inhibit AF synthesis was the same as that of the original lentinan, whereas their inhibition activity was not reduced at the increasing concentration. RT-PCR analyses were conducted to understand the effects of lentinan and its carboxymethylated derivatives on the transcription of certain genes associated with AF biosynthesis. The results showed that lentinan delayed the transcription of aflQ, whereas its carboxymethylated derivatives promoted the transcriptions of all the tested genes. Our results revealed that some chemical group features apart from the β-bond could play the vital role in the prevention of AF formation by polysaccharide, and highlighted the structural modifications which could promote its practicability in the control of aflatoxin contamination.