Nine independent experiments of NO3 − denitrification were analysed using the Arrhenius law and the Eyring’s transition-state theory to highlight how temperature affects reaction rate constants, affinities, and kinetic isotopic effects. For temperatures between 20 and 35 °C, the Arrhenius law and the transition-state theory described equally well observed temperature increases in 14NO3 − and 15NO3 −denitrification rates (R > 0.99 and residuals NRMSE < 3.39 %, p < 0.01). These increases were partly caused by an increase in frequency factor and a slight decrease in activation energy (enthalpy and entropy). Parametric analysis also showed that the affinity of 14NO3 − and 15NO3 − toward a microbial enzyme increased exponentially with temperature and a strong correlation with the rate constants was found (R = 0.93, p < 0.01). Experimental time- and temperature-averaged fractionation factor α P/S showed only a slight increase with increasing temperature (i.e. lower isotopic effects); however, a comprehensive sensitivity analysis in the concentration-temperature domain using average thermodynamic quantities estimated here showed a more complex response; α P/S was relatively constant for initial bulk concentrations [NO3 −]0 ≤ 0.01 mol kg−1, while substantial nonlinearities developed for [NO3 −]0 ≥ 0.01 mol kg−1 and appeared to be strongly correlated with microbial biomass, whose concentration and activity varied primarily as a function of temperature and available substrate. Values of α P/S ranging between 0.9 and 0.98 for the tested temperatures suggested that interpretations of environmental isotopic signatures should include a sensitivity analysis to the temperature as this affects directly the rate constants and affinities in biochemical reactions and may hide process- and source-related isotopic effects.