The RNA helicase DHX33 has been shown to be a critical regulator of cell proliferation and growth. However, the underlying mechanisms behind DHX33 function remain incompletely understood. We present original evidence in multiple cell lines that DHX33 transcriptionally controls the expression of genes involved in cell cycle, notably cyclins, E2F1, CDCs and MCMs. DHX33 physically associates with the promoters of these genes and controls the loading of active RNA polymerase II onto these promoters. DHX33 deficiency abrogates cell cycle progression, DNA replication, and leads to cell apoptosis. In zebra fish, CRISPR-mediated knockout of DHX33 results in downregulation of cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1 and MCMs in DHX33 knockout embryos. Additionally, we found the overexpression of DHX33 in a subset of non-small cell lung cancers and in Ras-mutated human lung cancer cell lines. Forced reduction of DHX33 in these cancer cells abolished tumor formation in vivo Our study demonstrates for the first time that DHX33 acts as a direct transcriptional regulator to promote cell cycle progression and plays an important role in driving cell proliferation during both embryo development and tumorigenesis.