PurposeThe present study aimed to explore the anticancer activity of hirsuteine (HST), an indole alkaloid from the traditional Chinese herbal medicineUncaria rhynchophylla, against colorectal cancer (CRC) and the underlining mechanism.MethodsMTT, colony formation, flow cytometry and MDC staining were conducted to confirm the antiproliferative effect of HST on human CRC cells harboring different p53 status. Protein expressions were evaluated by the Western blot analysis. p53 protein half-life and the interaction between p53 and MDM2 were investigated using cycloheximide (CHX)-chase assay and Co-immunoprecipitation (Co-IP), respectively. Transcriptional activity of p53 was examined by qRT-PCR and Chromatin immunoprecipitation (ChIP). Xenograft tumor in nude mice was created to evaluate in vivo anticancer effect of HST against CRC.ResultsHST inhibited cell growth, arrested cell cycle and induced autophagy, showing efficient anticancer effects on CRC cells independent of p53 status. In HCT-8 cells, HST prolonged wtp53 half-life, and upregulated mRNA level of p21, suggesting that HST activated the p53 pathway through enhancement of wtp53 stability and transcriptional activity. Meanwhile in SW620 cells, HST induced MDM2-mediated proteasomal degradation of mutp53R273H, increased the DNA-binding ability of mutp53R273Hat the p21 promoter, and upregulated mRNA levels of p21 and MDM2, demonstrating the depletion of mutp53R273Hand restoration of its wild-type-like properties by HST. p53 knockdown by siRNA significantly impaired the growth inhibition of HST on HCT-8 and SW620 cells. Moreover, HST showed anticancer effects in xenograft tumors, accompanied with an opposite regulation of wtp53 and mutp53R273Hin mechanism.ConclusionThis study revealed the anticancer efficacy of HST against CRC via opposite modulation of wtp53 and mutp53R273H, indicating the potential of HST to be a CRC drug candidate targeting p53 signaling.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12672-023-00688-1.