Microorganisms dominate the decomposition of organic matter and their activities are strongly influenced by temperature. As the carbon (C) flux from soil to the atmosphere due to microbial activity is substantial, understanding temperature relationships of microbial processes is critical. It has been shown that microbial temperature relationships in soil correlate with the climate, and microorganisms in field experiments become more warm-tolerant in response to chronic warming. It is also known that microbial temperature relationships reflect the seasons in aquatic ecosystems, but to date this has not been investigated in soil. Although climate change predictions suggest that temperatures will be mostly affected during winter in temperate ecosystems, no assessments exist of the responses of microbial temperature relationships to winter warming. We investigated the responses of the temperature relationships of bacterial growth, fungal growth, and respiration in a temperate grassland to seasonal change, and to 2 years’ winter warming. The warming treatments increased winter soil temperatures by 5–6°C, corresponding to 3°C warming of the mean annual temperature. Microbial temperature relationships and temperature sensitivities (Q10) could be accurately established, but did not respond to winter warming or to seasonal temperature change, despite significant shifts in the microbial community structure. The lack of response to winter warming that we demonstrate, and the strong response to chronic warming treatments previously shown, together suggest that it is the peak annual soil temperature that influences the microbial temperature relationships, and that temperatures during colder seasons will have little impact. Thus, mean annual temperatures are poor predictors for microbial temperature relationships. Instead, the intensity of summer heat-spells in temperate systems is likely to shape the microbial temperature relationships that govern the soil-atmosphere C exchange.