Significance We here describe the essential roles of RB and MRPS18-2 (S18-2) in homeostasis of cell stemness. Mouse primary cells expressing both S18-2 and RB (designated as RH18RB) exhibited a stem cell phenotype. As a proof of principle, we demonstrated decreased expression of stem-cell–related genes in human mesenchymal stem cells upon down-regulation of S18-2 and RB. Notably, loss of the S18-2 protein resulted in embryonic lethality in zebrafish. To reveal putative molecular mechanisms, we demonstrated that S18-2, in a multimeric complex with RB, enhanced the E3-ubiquitin ligase activity of RNF2 toward histone H2A. Finally, based on our observation that RH18RB cells generated tumors in severe combined immunodeficiency mice, S18-2 could be a promising target in the development of cancer therapies.