BackgroundGiardia duodenalis(referred to asGiardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide.Giardiacan be infected byGiardiavirus(GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV andGiardiavirulence is yet to be elucidated.MethodsTo identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization inGiardia trophozoiteswere examined by using Duolink proximal ligation assay (Duolink PLA).ResultsFrom Y2H screen, theGiardiachaperone protein,GiardiaDnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp inGiardia trophozoiteswere confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation ofGiardia.ConclusionTaken together, our results suggested a potential role of GdDnaJ in regulatingGiardiaproliferation and GLV replication through interaction with GLV RdRp.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05787-0.