Polychlorinated biphenyls (PCBs) are a group of persistent organochlorine compounds that have the potential to disrupt the homeostasis of thyroid hormones (THs) in fish, particularly juveniles. In this study, thyroid histology, plasma TH levels, and iodothyronine deiodinase (IDs, including ID1, ID2, and ID3) gene expression patterns were examined in juvenile Japanese flounder (Paralichthys olivaceus) following 25- and 50-day waterborne exposure to environmentally relevant concentrations of a commercial PCB mixture, Aroclor 1254 (10, 100, and 1000 ng/L) with two-thirds of the test solutions renewed daily. The results showed that exposure to Aroclor 1254 for 50 d increased follicular cell height, colloid depletion, and hyperplasia. In particular, hypothyroidism, which was induced by the administration of 1000 ng/L Aroclor 1254, significantly decreased plasma TT4, TT3, and FT3 levels. Profiles of the changes in mRNA expression levels of IDs were observed in the liver and kidney after 25 and 50 d PCB exposure, which might be associated with a reduction in plasma THs levels. The expression level of ID2 mRNA in the liver exhibited a dose-dependent increase, indicating that this ID isotype might serve as sensitive and stable indicator for thyroid-disrupting chemical (TDC) exposure. Overall, our study confirmed that environmentally relevant concentrations of Aroclor 1254 cause significant thyroid disruption, with juvenile Japanese flounder being suitable candidates for use in TDC studies.