Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by their activity of cleaving ubiquitin from specific protein substrates. Ubiquitin-Specific Protease 46 (USP46) has recently been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with a lysine codon (Lys 92) deletion in USP46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. However, whether this deletion affects enzyme activity is unknown. Here we show that USP46 has deubiquitinating enzyme activity detected by USP cleavage assay using GST-Ub52 as a model substrate. Interestingly, compared to wild type, the Lys 92 deletion mutant resulted in a decreased deubiquitinating enzyme activity of 27.04%. We also determined the relative expression levels of Usp46 in rat tissues using real-time RT-PCR. Usp46 mRNA was expressed in various tissues examined including brain, with the highest expression in spleen. In addition, like rat USP46, both human and mouse USP46 are active toward to the model substrate, indicating the USP cleavage assay is a simple method for testing the deubiquitinating enzyme activity of USP46. These results suggest that the Lys 92 deletion of USP46 could influence enzyme activity and thereby provide a molecular clue how the enzyme regulating the pathogenesis of mental illnesses.