Background. Sindbis virus (SV) is the prototype of alphaviruses which are a group of widely distributed human and animal pathogens. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein that shuttles between the nucleus and the cytoplasm. Our recent studies found that hnRNP A1 relocates from nucleus to cytoplasm in Sindbis virus (SV)-infected cells. hnRNP A1 binds to the 5' UTR of SV RNA and facilitates the viral RNA replication and translation. Methods. Making use of standard molecular techniques, virology methods and an in vitro system developed by our lab to assess the role of hnRNP A1 in SV positive strand RNA synthesis. Results. hnRNP A1 interacted with the genomic (G) and subgenomic (SG) RNA promoters. Knockdown of hnRNP A1 resulted in markedly decrease in the synthesis of G and SG RNA both in infected cells and in vitro. Conclusions. Our study provides the first direct evidence that hnRNP A1 actively participates in viral RNA replication and is required for the synthesis of G and SG RNA.