Zuotai, a famous Tibetan medicinal mixture containing beta-HgS, has been used to combine with herbal remedies for treating diseases for more than 1 300 years. The target organ for inorganic mercury toxicity is generally considered to be the kidney. Therefore, it is crucial to reveal the chemical speciation, spatial distribution and potential nephrotoxicity of mercury from Zuotai in kidney. To date, this remains poorly understood. We used Xray absorption spectroscopy (XAS) and micro X-ray fluorescence (mu-XRF) imaging based on synchrotron radiation to study mercury chemical forms and mercury special distribution in kidney after mice were treated orally with Zuotai, beta-HgS or HgCl2. Meanwhile, the histopathology of kidney was observed. Mice exposed with Zuotai showed kidney with significant proportion of mercury ions bound to sulfydryl biomolecules (e.g. Cys-S-lig-S-Cys) plus some of unknown species, but without methylmercury cysteine, which is the same as beta-HgS and HgCl2. The mercury is mainly deposited in renal cortex in mouse treated with Zuotai, beta-HgS or HgCl2, but with a low level of mercury in medulla. The total mercury in kidney of mice treated with HgCl2 was much higher than that of beta-HgS, and the later was higher than that of Zuotai. And, HgCl2 cause severe impairments in mouse kidney, but that was not observed in the Zuotai and beta-HgS groups. Meanwhile, the bio-metals (Ca, Zn, Fe and Cu) micro distributions in kidney were also revealed. These findings elucidated the chemical nature, spatial distribution and toxicity difference of mercury from Zuotai, beta-HgS and HgCl2 in mouse kidney, and provide new insights into the appropriate methods for biological monitoring.