Arsenic (As) is extremely toxic to living organisms at high concentrations. Arsenobetaine (AsB), confirmed to be a non-toxic form, is the main contributor to As in the muscle tissue of marine fish. However, few studies have investigated the biotransformation and biodegradation of AsB in mammals. In the current study, C57BL/6J mice were fed four different diets, namely, Yangjiang and Zhanjiang fish diets spiked with marine fish muscle containing AsB, and arsenite (As(III)) and arsenate (As(V)) diets spiked with As(III) and As(V), respectively, to investigate the biotransformation and bioaccumulation of AsB in mouse tissues for 42 d. Different diets exhibited different As species distributions, which contributed to varying levels of As bioaccumulation in different tissues. The intestines accumulated the highest level of As, regardless of form, which played a major part in As absorption and distribution in mice. We observed a significant biotransformation of AsB to As(V) following its diet exposure, and the liver, lungs, and spleen of AsB-treated mice showed higher As accumulation levels than those of As(III)- or As(V)-treated mice. Inorganic As showed relatively high accumulation levels in the lungs and spleen after long-term exposure to AsB. Overall, these findings provided strong evidence that AsB undergoes biotransformation to As(V) in mammals, indicating the potential health risk associated with long-term AsB intake in mammals.