Background: Aldehyde dehydrogenase (aldh) are a group of NAD(P)+-dependent enzymes involved in the biosynthesis of retinoic acid and a wide-range of metabolism. We previously demonstrated that inhibition of aldh activity with diethylaminobenzaldehyde (DEAB) expanded primitive hematopoeisis in zebrafish embryos and conducted a morpholino (MO)-mediated reverse genetic screen on the hematopoietic function of 15 individual aldh isoforms. Although none of the individual knock-down recapitulated DEAB treatment, we identified that aldh16a1, an uncharacterized isoform, may play a novel role in regulating erythropoiesis. Methods: Expression pattern of zebrafish aldh16a1 was examined by RT-PCR and whole-mount in situ hybridization (WISH). aldh16a1 knock-out model was generated with TALEN and effect on embryonic hematopoiesis was examined. ALDH16A1 was also knock-down using shRNA in human cord blood (CB) CD34+ population and the effect on hematopoietic lineage differentiation was examined by colony-forming assay. Results: aldh16a1 expressed along the mesoderm at early stages and predominantly in developing head and anterior pronephric duct at 24 hpf. Later at 48 hpf, it also expressed in causal hematopoietic tissue. TALEN-mediated aldh16a1 mutagenesis was confirmed by RFLP assay and DNA sequencing. In aldh16a1 mutant embryos, although early initiation of erythropoiesis as shown by gata1 expression at 12 hpf was not affected, erythropoiesis at 18 hpf was significantly reduced. Other hematopoietic lineages were not affected. TUNEL assay showed that apoptosis was induced in erythroid cells and erythroid maturation was also affected as shown by cell morphology. In human CB CD34+ population, knock-down of ALDH16A1 significantly reduced the number of CFU-E without affecting CFU-GEMM/GM. Conclusion: aldh16a1 knock-out significantly suppressed erythropoiesis by inducing apoptosis and also perturbing erythroid maturation. Known-down of ALDH16A1 in human CB CD34+ population also perturbed erythroid lineage differentiation, suggesting that Aldh16a1 may have a conserved role in regulating vertebrate erythropoiesis. Further studies are warrant to investigate the unknown underlying mechanism of Aldh16a1