Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Legumain activity has been detected in various mouse tissues including the kidney, spleen and epididymis. Legumain is overexpressed in the majority of human solid tumors and transcription of the legumain gene is regulated by the p53 tumor suppressor in HCT116 cells. The legumain activity is also increased under acid conditions in Alzheimer's disease brains. DJ-1/PARK7, a cancer- and Parkinson's disease-associated protein, works as a coactivator to various transcription factors, including the androgen receptor, p53, PSF, Nrf2, SREBP and RREB1. Recently, we found that legumain expression, activation and cleavage of annexin A2 are regulated by DJ-1 through p53. In this study, we found that the expression levels of legumain mRNA were increased in the cerebrum, kidney, spleen, heart, lung, epididymis, stomach, small intestine and pancreas from DJ-1-knockout mice, although legumain activity levels were decreased in the cerebrum, spleen and heart from DJ-1-knockout mice. Furthermore, we found that cystatin E/M expression was increased in the spleen, cerebrum and heart from DJ-1-knockout mice. These results suggest that reduction of legumain activity is caused by an increase of cystatin E/M expression in the spleen, cerebrum and heart from DJ-1-knockout mice.