Aristolochic acid I (AA-I) is a strong nephrotoxin, carcinogen, and mutagen found in plants such as the Aristolochia species. The mechanisms underlying AA-I toxicity in the kidneys are poorly understood. In this study, we aimed to gain insight into the mechanism of AA-I nephrotoxicity by analyzing the uptake, subcellular distribution, and intracellular targets of AA-I in the human kidney cell line HK-2 using immunocytochemistry, immunoprecipitation, and LC-MS/MS. In HK-2 cells incubated with 20[Formula: see text][Formula: see text]g/mL AA-I for different periods of time (up to 12[Formula: see text]h), AA-I was detected by a specific monoclonal antibody (MAb) against AA-I, both in the cytoplasm and nuclei. Nuclear localization depended on the exposure time. A protein with the molecular weight of 100 kDa was immunoprecipitated with the anti-AA-I MAb from the AA-I-treated cell lysates and was identified by LC-MS/MS as [Formula: see text]-actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti-[Formula: see text]-actinin-4 MAb. This evidence shows, for the first time, that [Formula: see text]-actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between [Formula: see text]-actinin-4 and AA-I nephrotoxic activity.