Supramolecular nanofibers based on D-amino acid-containing peptide self-assembly have attracted increased interest recently because of their superior biostability. However, the construction of nanostructures containing D-amino acids is still insufficient at present, and their application in biomedical fields should be further explored. Herein, we demonstrated that the aggregation property of hydrophobic anticancer tyroservaltide (YSV) could be decreased by introducing D-amino acids. Using such strategy, we fabricated a novel anticancer hydrogelator based on the D/L-peptide of NapGDFDFDYGYSV (D-YSV). Through a heating-cooling process, NapGDFDFDYGYSV self-assembled into a stable hydrogel, while its control peptide of NapGFFYGYSV (L-YSV) self-assembled into an unstable suspension. The nanofiber of D-YSV exhibited an excellent resistance to proteinase digestion comparing with that of L-YSV, and a better anticancer efficiency in vitro due to its improved cellular uptake. Moreover, D-YSV possessed good biocompatibility and showed a prominent tumour inhibition capacity in vivo via tail vein injection. Our study demonstrates a powerful strategy to reduce the aggregation property and construct supramolecular hydrogels of bioactive hydrophobic L-peptides.