Abstract: Approximately 25% of cases of Diamond Blackfan Anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40S ribosomal subunits. Rps19 is an essential protein which is strictly required for maturation of the 3'-end of 18S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1 and Rio2. When introduced in yeast Rps19, amino-acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells underexpressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40S particles and demonstrate for the first time the effect of Diamond Blackfan anemia associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.