Decapod iridescent virus 1 (DIV1) is a newly defined iridescent virus that infects decapod crustaceans and causes significant economic loss in aquaculture. To study the mechanisms of viral infection, an in vitro infection system is required. Here, we investigated the infection and replication of DIV1 in primary cultured hematopoietic tissue (HPT) cells of crayfish Cherax quadricarinatus. RT-PCR and Western blot analysis showed that DIV1 could infect the in vitro cultured cells and express viral genes. Immunofluorescent analysis of DIV1 major capsid protein indicated an infection rate of ∼23%. As the infection progressed, viral DNA copy numbers in both the cells and culture medium increased. Transmission electron microscopy analysis and animal challenging experiments demonstrated the production and release of infectious progeny virions. In summary, the primary culture of C. quadricarinatus HPT cells supports the infection and proliferation of DIV1. Thus, it can be used as an in vitro system to study the virus. •We investigate the replication of DIV1 in primary cultured HPT cells of Cherax quadricarinatus.•The in vitro cultured HPT cells support DIV1 proliferation.•The infection rate of DIV1 in cultured HPT cells is about 23%.•We provide an in vitro cell model for the study of DIV1.