During the last decade, aquaculture of the Pacific bluefin tuna (Thunnus orientalis) has been rapidly growing. At present, the most common form of the tuna farming is based on capturing wild juveniles. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, bluefin tuna-farming has succeeded in completing the reproduction cycle, but production bottlenecks remain to be solved because of very little biological information on the bluefin tuna. A compilation of genomic information on the bluefin tuna and development of high-throughput analytical methods promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Recently, our research group has reported a whole genome shotgun sequencing (WGS) of the bluefin tuna. Here, based on the WGS and newly sequenced large-scale expressed sequence tags (ESTs) data, we developed a 44K bluefin tuna oligonucleotide microarray and conducted an initial microarray experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as LPS, PHA or poly I:C. This microarray analysis identified specifically regulated genes for each treatment, including several known immune-related genes. Thus, this new 44K bluefin tuna oligonucleotide microarray successfully addressed a large number of immune pathways in parallel at the gene expression level. We expect that this microarray will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as of reproduction, development and influence of nutrition on tuna aquaculture production.