Arsenate [As(V)] is the main form of arsenic (As) present in freshwater taken up by freshwater fish. Data on the main uptake tissue, biotransformation, and bioaccumulation in freshwater fish exposed to As(V) were limited, and the reasons for its bioaccumulation in the muscle tissue of freshwater fish remain undetermined. Accordingly, we simulated bioaccumulation and depuration in zebrafish (Danio rerio) exposed to waterborne As(V) by employing a six-compartment physiologically based pharmacokinetic model and As speciation analysis. Modeling and biotransformation suggested that intestines were the main uptake site for waterborne As(V), instead of the gills. This novel finding was evidenced by the higher As transfer constant from water to intestines (k03 = 1.52 × 10-4 L d-1) compared to gills (k02 = 5.28 × 10-5 L d-1). The low concentration and percentage of arsenobetaine (AsB) in the intestines suggested a weak ability to synthesize AsB. Our results showed a substantial proportion of inorganic As in intestines and a relatively substantial percentage in muscle tissue. Therefore, high As(V) uptake in the intestines and lack of biotransformation contributed to high bioaccumulation of inorganic As in freshwater fish. Inorganic As posed concerns due to the human health risks associated with consuming As(V)-contaminated fish and should be addressed.