RNA helicases are a large family of proteins that are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). Every step of cellular RNA metabolism involves the activity of RNA helicases. Not surprisingly, more and more RNA helicases are reported to participate in the replication of viruses including the human immunodeficiency virus type 1 (HIV-1). Here, we provide evidence that overexpression of an RNA helicase named DHX30 enhances HIV-1 gene expression, but leads to the generation of viruses that package significantly low levels of viral RNA and exhibit severely decreased infectivity. These data reveal the complex roles of DHX30 in HIV-1 replication and implicate an inhibitory activity of DHX30 in HIV-1 RNA packaging.