This study was performed to elucidate the anti-proliferative effects and the apoptotic mechanisms of extracts from Lethariella zahlbruckneri in HT-29 human colon cancer cells. Both the acetone extract (AEL) and methanolic extract (MEL) of L. zahlbruckneri decreased viable cell numbers in a dose- and time-dependent manner in HT-29 cells. The AEL showed stronger cytotoxicity than MEL. Cell death induced by AEL increased cell populations in the sub-G1 phase, as well as the formation of apoptotic bodies and nuclear condensation, whereas MEL did not. Therefore, the potential of AEL to induce apoptosis was examined. Apoptosis induced by AEL was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. AEL stimulated Bid cleavage. This indicated that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. AEL increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. AEL also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that AEL inhibited HT-29 cell proliferation by inducing apoptosis, which might be mediated via both caspase-dependent and -independent pathways.