Mineralization of organic N sources by extracellular proteases affects both the availability of inorganic N to plants and losses of N to the environment. We hypothesized that (i) application of purified protease inhibitors would slow down soil N mineralization, and (ii) elevated concentrations of protease inhibitors in plant residues would also reduce soil and plant residue N mineralization. Our laboratory incubation studies showed that soil N mineralization was affected when soils were amended directly with specific inhibitors of different proteases. Isogenic controls and transgenic plants of Brassica (Brassica napus L.), Japonicum rice (Oryza sativa L.), and tobacco (Nicotiana tabaccum L.) showing enhanced wound-inducible protease inhibitor production did not differ in N concentration, C/N ratio, or lignin concentration in shoot residues, but protease inhibitor concentration was 1.5 to 2.3 times greater in the transgenic lines. In laboratory incubations with a loamy sand soil, inorganic N concentrations in leachate from transgenic plants were significantly less than isogenic controls for the first 30 d when the residues remained on the soil surface, then were higher at one or more dates thereafter. When residues were mixed with soil, differences were observed only for Brassica. In general, transgenic residues mineralized between 22 to 27% less N than their isogenic control plant residues in the first 30 d. However, no differences in soil N mineralization were detected between transgenic and isogenic residue treatments. Thus, protease inhibitors show potential for regulating short-term mineralization of N and protease inhibitor concentration in plant residues should be included with measures of total N concentration, C/N ratio, and lignin concentration to improve prediction of short-term N mineralization from plant residues.