D-limonene is recognized as a potential chemotherapeutic agent, however, the details of this mechanism remain unclear. In this study, we investigated the effects of d-limonene on colon cancer cell viability and its potential mechanism of action in vitro. After 48 h of treatment, d-limonene suppressed the viability of LS174T cells in a dose-dependent manner and caused a dose-dependent apoptotic cell death. D-limonene activated caspase-3 and -9 and PARP cleavage in a dose-dependent manner. Moreover, an increase in Bax protein and cytosol cytochrome c from mitochondria and a decrease in bcl-2 protein were observed following treatment with d-limonene. In addition, d-limonene decreased the levels of p-Akt (Ser473), p-Akt (Thr308) and p-GSK-3β (Ser9), suggesting that d-limonene induced apoptosis via the mitochondrial death pathway and the suppression of the PI3K/Akt pathway.