Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germline mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in two large DBA families. After filtering, one nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29) gene was present in all 5 DBA affected individuals and the obligate carrier, and absent from the unaffected non-carrier parent in one DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious, and resulted in haploinsufficiency of RPS29 expression compared with wildtype RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-rRNA processing defect compared to the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebrafish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for ribosomal RNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germline mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebrafish model.