A series of novel camphor-based pyrimidine derivatives (3a-3x) have been synthesized; their structures were determined by using conventional methods and compound 3f was further confirmed through single crystal XRD analysis. The cytotoxic activity of the target compounds against a panel of human normal (GES-1) and cancer cell lines (MDA-MB-231, RPMI-8226, A549) was evaluated by MTS assay. Here we found that compound 3f exhibited the strongest anti-tumor activity, comparable to that of etoposide, and had much lower cytotoxicity to normal GES-1 cells (IC > 50 μM) than the reference drug (IC = 8.89 μM). Subsequent mechanism studies in MDA-MB-231 cells revealed that compound 3f caused G0/G1 phase arrest and apoptosis in a dose dependent manner. Moreover, the loss of mitochondrial membrane potential and enhancement of cellular ROS levels were also observed upon 3f treatment, which indicated that 3f exerted cytotoxic activity by a ROS-mediated mitochondrial apoptosis pathway. This result was confirmed by a significant increase in the expression of pro-apoptotic proteins Bax, cytochrome C and caspase-3, and downregulation of anti-apoptosis protein Bcl-2. Overall, 3f can be adopted for further investigation in the development of antitumor agents based on natural products.