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Cystatin B was first described as an inhibitor of the cysteine family of proteases. Its biochemical properties have been studied in depth, the crystal structure of the protein has been determined and its enzymatic activity characterized. However, evidence from the literature suggests that cystatin B has alternative functions in addition to the inhibition of proteases. In this review we will discuss the results obtained from a number of laboratories, which detail such novel biological roles for cystatin B. In particular, we will discuss some of the pathologies involving the absence or presence of the protein. We will briefly summarize the function and structure of the protein, its possible involvement in tumoural growth and apoptosis, neurodegenerative diseases and immune response. The expression and cellular localization of cystatin B will be analyzed in different cell types and in developing rat cerebellum. The pattern will be matched to the expression of newly identified proteins that specifically interact with cystatin B. On the basis of all the experimental evidence we aim to propose a cytoplasmic function for cystatin B not necessarily related to its anti-protease activity.