Objective  Five types of nucleobase-alanines were synthesized to study their cytotoxicity and mechanism.  Methods  Five types of nucleobase-alanines were synthesized by the method  of nucleophilic ring-opening of serine-lactone. MTT assay was used to detect their effects on the viability of rat normal hepatocytes BRL-3A and rat hepatoma cells RH35. Flow cytometry was used to detect their effects of cell cycle and apoptosis on BLR-3A and RH35. Real-time PCR was performed to detect their effects of proliferation/apoptosis-related gene expression on these two cells.   Results  Five types of nucleobase-alanines were synthesized, including 1-uracil-L-alanine, 1-thymine-L-alanine, 1-cytosine-L-alanine, 9-adenine-L-alanine and 9-guanine-L-alanine. Among them, 1-cytosine-L-alanine and 9-guanine-L-alanine had significant lower half inhibition concentration(IC₅₀) values for RH35 than for BRL-3A, and their effects of promoting RH35 apoptosis were significantly stronger than that of BRL-3A, the amplitude of their promoting RH35 up-regulation apoptosis-related genes CASP3, CASP9 and Bax, the down-regulation of cell proliferation-related genes CCNA2 and PCAN, as well as the down-regulation of anti-apoptosis-related gene Bcl-2 were significantly higher than those of BRL-3A. Conclusion 1-cytosine-L-alanine and 9-guanine-L-alanine showed significant effects of inhibiting the viability and promoting apoptosis by up-regulating the expression of proliferation-related genes and down-regulating the expression of apoptosis-related genes more potently.