This study aimed to investigate the interaction between the ion-complementary self-assembling peptide RADA16-I and the hydrophobic drug mangiferin (MA), and the potential of the self-assembling peptide to be exploited as a drug carrier of MA.The RADA16-I-MA suspension was prepared by magnetic stirring, followed by fluorescence spectrophotometry, particle size determination, rheological properties analysis, and in vitro release assay to characterize the interaction between RADA16-I and MA. Then, the effects of in situ MA-loaded hydrogel on the proliferation of KYSE 30 and DLD-1 tumor cells and the toxic effect of the hydrogel on 293T renal epithelial cells were studied by the Cell Counting Kit 8 method.The RADA16-I-MA suspension was formed in water under magnetic stirring; the in situ hydrogel was formed when the suspension was added to PBS. The particle size in the RADA16-I-MA suspension was around 300-600 nm with an average size of 492 nm. Within 24 h, the cumulative release of MA from the RADA16-I-MA hydrogel was about 80%. The release rate of MA from the hydrogel was dependent on the concentration of RADA16-I and the release can be fitted with a first-order kinetic equation. The results suggested that the self-assembling peptide can stabilize MA in water to form a relatively stable suspension; the results also indicated that controlled release of MA from the RADA16-I-MA in situ hydrogel formed from the RADA16-I-MA suspension can be achieved by adjusting the concentration of the peptide in suspension. The cell viability studies showed that the RADA16-I-MA in situ hydrogel not only can maintain or enhance the intrinsic proliferation inhibition effects of MA on tumor cells, but also can reduce the toxicity of MA to normal cells.The self-assembling peptide RADA16-I can be a potential candidate for constructing a delivery system of the hydrophobic drug MA.