Warming-induced increases in microbial CO2 release in northern tundra may positively feedback to climate change. However, shifts in microbial extracellular enzyme activities (EEAs) may alter the impacts of warming over the longer term. We investigated the in situ effects of 3 years of winter warming in combination with the in vitro effects of a rapid warming (6 days) on microbial CO2 release and EEAs in a subarctic tundra heath after snowmelt in spring. Winter warming did not change microbial CO2 release at ambient (10°C) or at rapidly increased temperatures, i.e., a warm spell (18°C) but induced changes (P < 0.1) in the Q10 of microbial respiration and an oxidative EEA. Thus, although warmer winters may induce legacy effects in microbial temperature acclimation, we found no evidence for changes in potential carbon mineralization after spring thaw.