There is active debate about whether respiratory processes of soil microorganisms will acclimate to elevated temperatures. If they do so this may provide a negative feedback to losses of soil carbon expected under a warmer climate. Temperature acclimation may arise physiologically but at the level of the soil microbial community it may also manifest via evolutionary adaptation and/or species turnover. We tested whether acclimation to elevated temperature occurred in response to long-term soil warming and seasonal variation in field temperatures. Soils were sampled from the Harvard Forest, long-term, soil warming experiment in April, July, August and November 2006. 13C-labeled substrates were added to soils in the laboratory and then incubated across 24 h at three different temperatures (10, 15 and 20°C). Mineralization rates of these substrates were determined through CO2 concentration determinations and isotope mixing models. Responses of organic substrate mineralization rates were dependent on the substrate considered, the time of sampling, the warming treatment and the incubation treatment. Exploration of three-way interactions between warming treatment × substrate × incubation temperature and sampling date × substrate × incubation temperature indicated both warming treatment- and seasonally-induced acclimation, respectively. Temperature optima for mineralization rates were higher in the warmed treatment and in warmer sampling months (when compared to the non-warmed controls and the cooler sampling months, respectively). These data suggest that long-term soil warming results in functionally different soil microbial communities, with distinct temperature optima and substrate utilization rates, and also that these communities functionally vary across the growing season.