Infections with the mosquito-borne dengue virus (DENV) remain a significant public health challenge. In the absence of a commercial therapeutic to treat DENV infection, a greater understanding of the processes of cellular replication is required. The abundant cellular chaperone protein heat shock protein 90 (Hsp90) has been shown to play a proviral role in the replication cycle of several viruses, predominantly through the stabilization of specific viral proteins. To investigate any potential role of Hsp90 in DENV infection the interaction between Hsp90 and DENV proteins was determined through co-immunoprecipitation experiments. Six DENV proteins namely envelope (E) and nonstructural (NS) proteins NS1, NS2B, NS3, NS4B and NS5 were shown to interact with Hsp90, and four of these proteins (E, NS1, NS3 and NS5) were shown to colocalize to a variable extent with Hsp90. Despite the extensive interactions between Hsp90 and DENV proteins, inhibition of the activity of Hsp90 had a relatively minor effect on DENV replication, with inhibition of Hsp90 resulting in a decrease of cellular E protein (but not nonstructural proteins) coupled with an increase of E protein in the medium and an increased virus titer. Collectively these results indicate that Hsp90 has a slight anti-viral effect in DENV infection.