Arsenic is ubiquitously present in the aquatic environment. We investigated the acute toxic effects of arsenite [As(III)] exposure on rare minnows (Gobiocypris rarus) in vivo. The 96-h LC50 value for exposure to As(III) was 13.73 mg/L. As(III) bioaccumulation in different tissues was measured using inductively-coupled plasma mass spectrometry, and the extent of As(III) accumulation was, from greatest to least, liver > intestine > gills > muscle > kidney > testis > brain. Histological examination revealed that in As(III)-treated fish, numerous cellular and tissue alterations were present in the gill, liver, and intestine tissues. Moreover, transmission electron microscopy showed ultrastructural alterations in hepatocytes. We also performed transcriptome analyses to investigate As(III)-induced toxicity response in the liver of As(III)-treated fish; various oxidative-related genes were differentially expressed, and their expression levels were further validated using qPCR. This study is one of the many steps we aim to take on the way to promote the rare minnow to an international standard laboratory animal.