We demonstrate, for the first time, that Singapore Grouper Iridovirus (SGIV) can successfully infect a Zebrafish cell line. Combined with the recent availability of the complete zebrafish (danio rerio) genome, this provides an opportunity to investigate virus-host interactions at the molecular level. Using iTRAQ labeling and two-dimensional LC/MS/MS quantitative proteomics, 157 zebrafish proteins exhibiting significant alterations in expression levels following SGIV infection were identified. Gene ontology analysis revealed that SGIV controls a wide aspect of zebrafish host machinery to ensure replication and propagation. In order to probe the mechanism underlying SGIV infection in Zebrafish cells, we used an anti-sense morpholino to knockdown orf86r, an immediate early viral gene that encodes the SGIV protein ORF86R. The expression profile of certain host proteins involved in replication was altered upon knockdown. In particular, expression of CNOT, a non-enzymatic subunit of the CCR4-NOT transcription complex was markedly affected. Taken together, these findings provide a new insight on the function of the essential viral protein ORF86R. Our results show that Singapore Grouper Iridovirus infection of a Zebrafish cell line is a useful new tool to study virus-host interactions.