Abstract.Hedyotis diffusa Willd (HDW) is a widely used traditional Chinese medicine in clinical therapy to treat various types of cancer, including colorectal cancer (CRC), but its effective polar fractions and functional mechanisms remain unclear. The aim of the present study was to determine the most effective extract of HDW and to investigate its effects on the regulation of CRC cell proliferation and apoptosis, as well as to investigate the underlying molecular mechanisms. The results demonstrated that the chloroform extract of HDW (CEHDW) exhibited the most anticancer ability. Furthermore, results of the MTT assay, colony formation, carboxyfluorescein diacetate succinimidyl ester assay and annexin V/propidium iodide staining suggested that CEHDW significantly inhibits proliferation and promotes apoptosis in the SW620 CRC cell line. Additionally, reverse transcription-polymerase chain reaction and western blot analysis demonstrated that CEHDW treatment downregulated the expression of Survivin, proliferating cell nuclear antigen, Cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma 2 (Bcl-2), and upregulated the expression of Bcl-2-associated X protein at the mRNA and protein levels. CEHDW also decreased the phosphorylation of protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK), which indicated that the suppression of the AKT and ERK signaling pathways may be one of the underlying molecular mechanisms by which CEHDW exhibited its anticancer effect. Thus, CEHDW may be a promising agent for anticancer therapy.