Arsenate is a pentavalent form of arsenic that shares similar chemical properties to phosphate. It has been shown to be taken up by phosphate transporters in both eukaryotic and prokaryotic microbes such as yeast and Escherichia coli. Recently, the arsenate uptake in vertebrate cells was reported to be facilitated by mammalian type II sodium/phosphate transporter with different affinities. As arsenate is the most common form of arsenic exposure in aquatic system, identifying the uptake pathway of arsenate into aquatic animals is a crucial step in the elucidation of the entire metabolic pathway of arsenic. In this study, the ability of a zebrafish phosphate transporter, NaPi-IIb1 (SLC34a2a), to transport arsenate was examined. Our results demonstrate that a type II phosphate transporter in zebrafish, NaPi-IIb1, can transport arsenate in vitro when expressed in Xenopus laevis oocytes. NaPi-IIb1 mediates a high-affinity arsenate transport, with a K(m) of 0.22 mM. The natural substrate of NaPi-IIb1, dibasic phosphate, inhibits arsenate transport. Arsenate transport via NaPi-IIb1 is coupled with Na(+) and exhibits sigmoidal kinetics with a Hill coefficient of 3.24 ± 0.19. Consistent with these in vitro studies, significant arsenate accumulation is observed in all examined zebrafish tissues where NaPi-IIb1 is expressed, particularly intestine, kidney, and eye, indicating that zebrafish NaPi-IIb1 is likely the transport protein that is responsible for arsenic accumulation in vivo.