DEAD-box helicase 41 (DDX41) is an RNA helicase and accumulating evidence has suggested that DDX41 is involved in pre-mRNA splicing during tumor development. However, the role of DDX41 in tumorigenesis remains unclear. In order to determine the function of DDX41, the human DDX41 gene was cloned and overexpressed in HeLa cells. The present study demonstrated that DDX41 overexpression inhibited proliferation and promoted apoptosis in HeLa cells. RNA-sequencing analysis of the transcriptomes in overexpressed and normal control samples. DDX41 regulated 959 differentially expressed genes compared with control cells. Expression levels of certain oncogenes were also regulated by DDX41. DDX41 selectively regulated the alternative splicing of genes in cancer-associated pathways including the EGFR and FGFR signaling pathways. DDX41 selectively upregulated the expression levels of five antigen processing and presentation genes (HSPA1A, HSPA1B, HSPA6, HLA-DMB and HLA-G) and downregulated other immune-response genes in HeLa cells. Additionally, DDX41-regulated oncogenes and antigen processing and presentation genes were associated with patient survival rates. Moreover, DDX41 expression was associated with immune infiltration in cervical and endocervical squamous cancer. The present findings showed that DDX41 regulated the cancer cell transcriptome at both the transcriptional and alternative splicing levels. The DDX41 regulatory network predicted the biological function of DDX41 in suppressing tumor cell growth and regulating cancer immunity, which may be important for developing anticancer therapeutics.