Unlike differentiated cells, cancer stem cells (CSCs) can generate malignancy. Conventional chemotherapy results in CSCs spreading causing metastasis and relapse. Differentiation is an emerging approach providing a safer strategy to eradicate CSCs. Jasonia montana is a medicinal plant that occurs in Sinai Peninsula in Egypt. Here, we used two human colorectal cancer cell lines; HCT-116 and Caco-2 to investigate the ability of methanolic and petroleum ether extracts of Jasonia montana to differentiate colon CSCs. Tandem Mass–Spectrometry (MS/MS) revealed that quercitrin and 5-Hydroxycostic acid represent the major extracts ingredients. Cytotoxicity assay (SRB) illustrated that petroleum ether extract inhibited Caco-2 cells (IC50=10 μg/ml), whereas, methanolic extract inhibited HCT-116 cells (IC50=20 μg/ml). On treating HCT-116 and Caco-2 cells with methanolic and petroleum ether extract respectively in diluted doses to alleviate their cytotoxicity, qRT-PCR showed a significant increase in CK20 expression and a significant decrease in CD44 expression, a significant decrease in Sox-2 and NOTCH-1 expression and a significant increase in PTEN expression. Furthermore, qRT–PCR showed a significant decrease in miR-21-5P and miR-10b-5P expression and significant increase in miR-34a-5P and miR-145 expression. Our results suggest, for the first time, that Jasonia montana methanolic and petroleum ether extracts can induce colorectal CSCs differentiation.