DEAD-box helicase 1 (DDX1) is a multifunction protein involved in diverse cellular processes including transcription, viral replication, mRNA/miRNA processing, and tRNA splicing. Here, we report a novel function of DDX1 in mRNA alternative splicing in pancreatic beta cells. By performing integrated data analysis of high-throughput RNA sequencing (RNA-Seq), and cross-linking and immunoprecipitation coupled with deep sequencing (CLIP-Seq), we identify hundreds of alternative splicing genes that are targeted by DDX1. These DDX1-targeted alternative splicing genes are mainly associated with calcium ion binding, high voltage-gated calcium channel, and transmembrane transporter. Functionally, silencing DDX1 impairs calcium influx and insulin secretion in the pancreatic beta cells. These results reveal an important role for DDX1 in the regulation of gene alternative splicing and insulin secretion in pancreatic beta cells. (C) 2018 Elsevier Inc. All rights reserved.