Lyn, a tyrosine kinase which is activated by double stranded DNA damaging agents, is involved in various signaling pathways such as proliferation, apoptosis and DNA repair. RpS3 is involved in protein biosynthesis as a component of the ribosome complex, and has an endonuclease activity to repair damaged DNA. In this report, we show that rpS3 and Lyn interact each other and the phosphorylation of rpS3 by Lyn, forming ribosome heterogeneity, appears to upregulate the translation of p-glycoprotein, a gene product of MDR1 (multi-drug resistance gene 1). Also, we found that two different regions of rpS3 protein were associated with the SH1 and SH3 domains of Lyn. In vitro immunocomplex kinase assay indicated that rpS3 protein acts as a substrate of Lyn which phosphorylates Y167 residue of rpS3. Furthermore, through the addition of various kinase inhibitors, we confirmed that the phosphorylation status of rpS3 is regulated by both Lyn and doxorubicin. We demonstrate that the phosphorylation of rpS3 by Lyn increases drug resistance of cells through the upregulation of p-glycoprotein translation.