ETHNOPHARMACOLOGICAL RELEVANCE: It is well known that Shaoyao Decoction (SYD), as a commonly used formula of traditional Chinese medicine (TCM) has a beneficial effect on the treatment of ulcerative colitis (UC). It is found that SYD can also prevent colitis-associated colorectal cancer (CAC). However, its potential anti-cancer mechanism is still waiting to be revealed. AIMS OF THE STUDY: The aim of this study is to investigate the underlying mechanisms of SYD in inhibiting CAC through silico analysis as well as animal experiment validation. MATERIALS AND METHODS: The primary active compounds, potential therapeutic targets and intervening signaling pathways, which SYD might inhibit the CAC process were predicted by network pharmacology analysis combined with our previous research result of high performance liquid chromatography (HPLC). We attempted to validate the acquired hub targets from molecular docking combined with the Gene Expression Profiling Interactive Analysis (GEPIA), the Human Protein Atlas (HPA), and the cBioPortal database comprehensively. Subsequently, an animal model of CAC mice induced by Azioximethane (AOM) and dextran sulfate sodium (DSS) was constructed and treated with SYD for 14 weeks, and tumor-related physical indicators were evaluated after sacrificed. In addition, samples of colon tissues were obtained for histologic and protein level studies to verify the predicted mechanism. RESULTS: We obtained 167 active ingredients of SYD and predicted 148 potential targets through network pharmacology analysis, among which quercetin, berberine, kaempferol, wogonin and naringenin were selected as core drug ingredients, and TP53, AKT1, CASP3, PTGS2 and CCND1 were identified and included into the range of core targets. GO and KEGG analyses suggested that the PI3K-Akt signaling pathway may hold a crucial role in CAC prevention and treatment by promoting apoptosis and inhibiting tumor proliferation. In the animal experiment, both SYD and SASP treatments improved the inflammatory condition and pathological damage of the colon in mice. After treatment with SYD and SASP, it was found that a decrease of Cyclin D1, and Survivin expression levels and increase of p53, Cleaved caspase-3 expression levels could be mediated by decreasing the phosphorylation levels of PI3K and Akt proteins in the colon tissues of mice. CONCLUSION: The results of our study provide supports that SYD effectively inhibits CAC based on modulating PI3K-Akt signaling pathway to suppress tumor proliferation process as well as to promote tumor apoptosis process.