Cadmium (Cd) is known to cause various disorders in the testis, and metallothionein (MT) is known as a protein, which has a detoxification function for heavy metals. However, the changes of Fe, Cu, and Zn distribution in the testis induced by Cd exposure have not been well examined. Moreover, only a few studies have been reported on the localization of MT after Cd exposure. In this study, we have investigated the changes of Fe, Cu, and Zn distribution in Cd-exposed testis by a newly developed in air micro-Particle Induced X-ray Emission (PIXE) method. Also, we examined the distribution of MT expression in testis. In the testis of Cd-treated rats with significant increases of lipid peroxidation, the sertoli cell tight junction was damaged by Cd exposure, resulting from disintegration of the blood testis barrier (BTB). Evaluation by in air micro-PIXE method revealed that Cd and Fe distribution were increased in the interstitial tissues and seminiferous tubules. The histological findings indicated that the testicular tissue damage was advanced, which may have been caused by Fe flowing into seminiferous tubules followed by disintegration of the BTB. As a result, Fe was considered to enhance the tissue damage caused by Cd exposure. MT was detected in spermatogonia, spermatocytes, and Sertoli's cells in the testis of Cd-treated rats, but was not detected in interstitial tissues. These results suggested that MT was induced by Cd in spermatogonia, spermatocytes, and Sertoli's cells, and was involved in the resistance to tissue damage induced by Cd.