Our study aimed to explore how DEK, a carcinogenic protein with chromatin architectural function, genome-widely binds to RNA and affects the alternative splicing in cancer cells to decipher its molecular functions. To achieve this goal, cell phenotype experiments, RNA sequencing (RNA-seq), and improved RNA immunoprecipitation sequencing (iRIP-seq) were conducted to identify the function and regulated targets of DEK in HeLa cells. The results showed DEK overexpression promoted cell proliferation and invasion of HeLa cells. Meanwhile, DEK hardly affected transcript level expression of those high expressed genes, but splicing pattern of 411 genes was regulated by DEK in HeLa cells, which were enriched in Hippo signaling pathway. Moreover, DEK broadly bind the RNA of a total of 11, 112 genes, with a biased binding the 5' splice site (5'SS) consensus GGUAA motifs at the CDS and intronic regions. In addition, 297 DEK-binding genes showed different splicing pattern after DEK overexpression in HeLa cells. These genes were enriched in Hippo signaling pathway including CSNK1D. The RT-qPCR and RIP-PCR confirmed that DEK can bind to CSNK1D to regulate its alternative splicing in HeLa cells. In summary, our results indicated DEK could broadly bind and regulate the pre-mRNA splicing process, which provide new insights of mechanisms that DEK functions in various biological processes including cancer.