Hydrothermal treatment improves the digestibility of pulses through the coupled phenomena of enzymatic degradation and diffusion of galacto-oligosaccharides, despite the thermal inactivation of -galactosidase at mild temperatures. To better understand the key process parameters that influence the balance between enzyme activity and its inactivation, the kinetics of -galactosidase inactivation were investigated in a cowpea-water system at a broad range of temperatures (45 °C 60 °C), moisture contents (0.16 g/g dry basis, d.b. 0.85 g/g d.b.) and treatment times (5 min 7.5 h). For moisture contents ranging from 0.25 to 0.85 g/g d.b., the higher the temperature the faster the inactivation of -galactosidase; while for 0.16 g/g d.b., the relative enzymatic inactivation was not temperature-dependent. A first-order model was successfully fitted to the experimental data, whereas the temperature dependence of inactivation rate followed the Arrhenius model. The influence of moisture content on the enzymatic inactivation rate was remarkable. The inactivation rate constant at excess water (0.85 g/g d.b.) was five times higher than the samples at limited water (0.35 g/g d.b.) condition. The results showed moisture-dependent -galactosidase inactivation kinetics mainly at mild temperatures (55 and 60 °C), with implications for the representation of diffusion-reaction phenomena during hydrothermal processing of pulses.