Seven new daphnane-type diterpenoids, daphgenkins A-G (<b>1</b>-<b>7</b>), and 15 known analogues (<b>8</b>-<b>22</b>) were isolated from the flower buds of <i>Daphne genkwa</i>. Their structures and absolute configurations were elucidated by spectroscopic data and calculated ECD analyses. The cytotoxicities of all daphnane-type diterpenoids (<b>1</b>-<b>22</b>) obtained were evaluated against three human colon cancer cell lines (SW620, RKO, and LoVo). Compounds <b>1</b>, <b>12</b>, and <b>13</b> exhibited cytotoxic effects against the SW620 and RKO cell lines, with IC<sub>50</sub> values in the range of 3.0-9.7 μM. The most active new compound, <b>1</b>, with an IC<sub>50</sub> value of 3.0 μM against SW620 cells, was evaluated further for its underlying molecular mechanism. Compound <b>1</b> induced G<sub>0</sub>/G<sub>1</sub> cell cycle arrest, leading to the induction of apoptosis in SW620 cells. Also, it induced cancer cell apoptosis by an increased ratio of Bax/Bcl-2, activated cleaved caspase-3 and caspase-9, and upregulated PARP. Finally, compound <b>1</b> significantly inhibited PI3K/Akt/mTOR signaling in SW620 cells. Together, the results suggest that compound <b>1</b> may be a suitable lead compound for further biological evaluation.