RFX6 transcription factor is believed to play a central role in directing cell development of insulin-producing pancreatic islet. RFX6 homozygous mutations cause syndromic neonatal diabetes with hypoplastic pancreas. However, RFX6 heterozygous mutations cause maturity-onset diabetes of the young (MODY) with normal pancreas development. Here, we show that RFX6 may control islet cell development and insulin production in different manners. The rfx6 knockout zebrafish generated by CRISPR/Cas9 exhibited an overt diabetes phenotype. Pancreatic islet failed to form compact structures in the knockout fish. While endocrine pancreatic islet non-β-cells were absent, insulin-producing β-cells were present in the knockout fish. Although insulin mRNA level was normal in the β-cells of the knockout fish, insulin protein level was decreased. High-throughput RNA sequencing (RNAseq) showed that differentially expressed genes were enriched in the translation term in islet β-cells from the knockout fish. Chromatin immunoprecipitation sequencing (ChIPseq) of normally developed islet β-cells from mice demonstrated that rfx6 interacted with translation initiation factors and controlled insulin translation. Our data indicate that Rfx6 may act as a transcription factor regulating the transcription of genes involved in mRNA translation, which may represent a new mechanism and treatment strategy for diseases.