RAD51 is involved in the homologous recombination of DNA double-strand breaks by being directed to single-stranded DNA with the assistance of the BRCA2 protein. Therefore, blocking the interaction between RAD51 and BRCA2 is considered to be a potential anticancer therapy. Currently, D-peptide inhibitors are widely recognized for their biological stability, low immunogenicity and target specificity. Here, we have identified a novel, potent and biostable d-amino acid-containing peptide inhibitor (RB-1) that blocks the RAD51-BRCA2 interaction through an integrated virtual screening protocol. MST and FP experiments showed that RB-1 had excellent binding affinity for RAD51. MD simulation confirmed the stable binding of RB-1 to the active binding site of RAD51. Furthermore, RB-1 exhibited significant antiproliferative activity on a panel of kidney cancer cell lines and less toxicity to normal cells, suggesting its potential therapeutic effects. Meanwhile, RB-1 exerted antitumor effects by inhibiting HR repair. In addition, RB-1 had good biological stability in mouse serum, highlighting its potential for in vivo activity. In vivo studies showed that RB-1 can effectively suppress tumor growth in mice without causing serious systemic side effects. In conclusion, these results suggest that d-amino acid-containing peptide RB-1 is a promising antitumor agent for kidney cancer and merits further investigation.