Abstract: Protein biosynthesis by the ribosome is a fundamental biological process in living systems. Recent studies suggest that ribosomal subunits also play essential roles in cell growth and differentiation beyond their roles in protein translation. The ribosomal subunit RPS6 has been studied for more than 50 years in various organisms, but little is known about its specific roles in certain signaling pathways. In this study, we focused on the functions of Arabidopsis RPS6A in auxin-related root growth and development. The rps6a mutant presented a series of auxin-deficient phenotypes, such as shortened primary roots, reduced lateral root numbers, and defective vasculatures. Treatment of the rps6a mutant with various concentrations of auxin and its analogs did not restore the root defect phenotypes, suggesting a defect in the auxin signaling pathway. Further cell biological and global transcriptome analyses revealed that auxin signaling was weakened in the rps6a mutant and that there was a reduced abundance of PIN-FORMED (PIN) auxin transporters. Our work provides insights into the role of the protein biosynthesis pathway involved in auxin signaling.