Isorhamnetin, a flavonoid isolated from the fruits of herbal medicinal plants, such as Hippophae rhamnoides L., exerts anticancer effects similar to other flavonoids. However, the effect of isorhamnetin on colorectal cancer (CRC) and the underlying molecular mechanism are unclear. This study aimed to determine the effect of isorhamnetin on the proliferation of cells from the human CRC cell lines, HT-29, HCT116 and SW480. It was demonstrated that isorhamnetin suppressed the proliferation of cells from all three cell lines, induced cell cycle arrest at the G2/M phase and suppressed cell proliferation by inhibiting the PI3K-Akt-mTOR pathway. Isorhamnetin also reduced the phosphorylation levels of Akt (ser473), phosph-p70S6 kinase and phosph-4E-BP1 (t37/46) protein, and enhanced the expression of Cyclin B1 protein. Therefore, this compound was revealed to be a selective PI3K-Akt-mTOR pathway inhibitor, and may be a potent anticancer agent for the treatment of CRC, as it restrains the proliferation of CRC cells.