Understanding the temperature sensitivity of soil respiration is critical for predicting the response of ecosystems to climate change, yet the microbial communities responsible are rarely considered explicitly in studies or models. In this study, we assessed total microbial community composition, quantified bacterial respiration temperature response, and investigated the temperature dependence of bacterial carbon substrate utilization in tropical, temperate, and taiga soils (from Puerto Rico, California, and Alaska). Microbial community composition was characterized using phospholipid fatty acid analysis. Bacterial community respiration on a standardized set of substrates was ascertained using the BiOLOG™ substrate utilization assay incubated at four temperatures: 4, 12, 28, and 40 °C. First, we found that microbial communities from the three latitudes were compositionally distinct and that the bacterial component of the three communities had markedly different respiration temperature–response curves corresponding with their experienced temperature regimes. We use these data to highlight limitations of widely used temperature–response equations and investigate temperature-dependent patterns of substrate utilization. We found that temperature response, in terms of both respiration rates and substrate use, varied for these bacterial communities independent of substrate quality or quantity interactions such as labile depletion. In contrast to the common assumption of heterotrophic microbial ubiquity, we found that bacterial community differences from these diverse systems appeared to determine both rates of respiration and patterns of carbon substrate usage. We suggest that microbial community composition-specific responses to changing climate may be important in predicting the long-term role of ecosystems in atmospheric CO2 dynamics.