Understanding the drivers of aerobic methane (CH 4 ) oxidation (MOX) is paramount in assessing the current and potential future CH 4 emissions from freshwater aquatic systems. Regulation of MOX kinetics is a complex function of CH 4 and oxygen (O 2 ) concentrations. While MOX activity is usually proportional to the concentration of CH 4 itself, the effects of O 2 have been more conflicting, with maximum MOX rates often restricted to low O 2 concentrations. Despite the complexity involved, MOX kinetics are often modelled as monotonic positive functions of both CH 4 and O 2 concentrations. We conducted a series of incubation experiments using natural and unamended water samples obtained from multiple depths in northern temperate lakes that vary widely and independently in their CH 4 and O 2 concentrations. Our results showed the expected positive effect of CH 4 concentration and temperature but also demonstrated the strong inhibitory effects of O 2 at high concentration. We then developed a general model describing the kinetics of MOX, simultaneously integrating the effects of CH 4 concentration, temperature as well as the non-linear effect O 2 on MOX activity. The model revealed an overall temperature dependency (activation energy = 0.49 ± 0.06 eV) much lower than reported for methanogenesis and an optimal O 2 level around 15 μmol O 2 L −1 where maximum MOX activity occurs, regardless of CH 4 concentration and temperature. We further show that ignoring the inhibitory effect of O 2 can lead to significant bias in calculating the expected MOX rates in different portions of the water column.