Abstract: Mitochondrial ribosomal proteins (MRPs) are a category of proteins that are regulated by both cell nucleus and mitochondria. In this study, we have observed a down-regulation of the mitochondrial ribosomal protein MRPS28 in non-small cell lung cancer A549 cells following exposure to carbon ion irradiation. Utilizing small interfering RNA (siRNA)-mediated gene silencing to specifically target MRPS28 in A549 cells, and employing the CRISPR/Cas9 system for genomic editing, we have successfully generated a stable A549 cell line with attenuated MRPS28 expression. Comparative analysis with the control group revealed that the down-regulation of MRPS28 expression resulted in a significant reduction in both the proliferative capacity and the survival rate of A549 cells. Simultaneously, the knockdown of MRPS28 leads to an increase in intracellular reactive oxygen species (ROS) content, a decrease in mitochondrial membrane potential, and the promotion of apoptosis. Upon employing Western Blot to evaluate the expression profiles of proteins implicated in apoptosis, it was observed that the down-regulation of MRPS28 induces apoptosis through the activation of the p53-dependent mitochondrial apoptotic cascade. Furthermore, in the A549 cell line, the combined effect of MRPS28 gene expression down-regulation and carbon ion irradiation significantly inhibited cellular proliferative activity and reduced survival capacity, while also accelerating the process of irradiation-induced apoptosis. These results suggest that down-regulation of MRPS28 expression may enhance the radio-sensitivity of A549 cells to carbon ion radiation. The present investigation provides initial evidence that down-regulation of MRPS28 contributes to apoptosis in A549 cells induced by carbon ion irradiation, thereby augmenting the radio-sensitivity of A549 cells to carbon ion.