In the present work, 103 novel acyclic nucleosides were designed, synthesized, and evaluated for their anticancer activities <i>in vitro</i> and <i>in vivo</i>. The structure-activity relationship (SAR) studies revealed that most target compounds inhibited the growth of colon cancer cells <i>in vitro</i>, of which 3-(6-chloro-9<i>H</i>-purin-9-yl)dodecan-1-ol (<b>9b</b>) exhibited the most potent effect against the HCT-116 and SW480 cells with IC<sub>50</sub> values of 0.89 and 1.15 μM, respectively. Furthermore, all of the (<i>R</i>)-configured acyclic nucleoside derivatives displayed more potent anticancer activity compared to their (<i>S</i>)-counterparts. Mechanistic studies revealed that compound <b>9b</b> triggered apoptosis in the cancer cell lines <i>via</i> depolarization of the mitochondrial membrane and effectively inhibited colony formation. Importantly, compound <b>9b</b> inhibited the growth of the SW480 xenograft in a mouse model with low systemic toxicity. These results indicated that acyclic nucleoside compounds are viable as potent and effective anticancer agents, and compound <b>9b</b> may serve as a promising lead compound that merits further attention in future anticancer drug discovery.