The priming effect on soil organic carbon (SOC) mineralization is closely related to carbon (C) sequestration. Pyrogenic organic matter (POM) has been proposed as an effective C sequestration material that might significantly contribute to the alleviation of climate change. However, the mechanisms involved in POM-induced priming effects are not well understood. We conducted a field microcosm study of the priming effects induced by rice straw POMs produced at 300 °C (P300) and 500 °C (P500) in a subtropical plantation (without roots, Alfisols, pH 6.4), and explored the involved mechanisms. The direction of the primed SOC mineralization exhibited three distinct stages as positive-negative-positive in the P300 treatment, while the opposite direction at each stage occurred in the P500 treatment during the entire 182 days’ experiment. Overall, the cumulative primed SOC mineralization was estimated to be 30.3 g C m−2 with a relative size of 22.3% in the P300 treatment, while it was −19.4 g C m−2 and −13.6% in the P500 treatment. At the end of the experiment, the P300 increased the soil microbial biomass and decreased the relative abundance of actinomycetes as well as Gram-negative to Gram-positive (G–/G+) bacteria ratio. However, it did not influence the examined extracellular enzyme activities except for the reduction of β-D-glucosidase activity, which suggested an apparent positive priming effect. On the contrary, the P500 decreased the soil microbial biomass and increased the relative abundance of actinomycetes as well as G–/G+ bacteria ratio, and increased phenol oxidase and peroxidase activities. This signified that soil microorganisms were inclined to decompose relatively recalcitrant organic C. Our 13C isotope analysis indicated that the P300 increased the soil-derived hot water-extractable C, while the P500 had no influence. Additionally, soil- and POM-derived carbon dioxides were positively correlated in both treatments. Therefore, these results collectively suggested that the P300-induced increase in SOC mineralization resulted from the activation of soil microorganisms. On the contrary, the P500-induced decrease in SOC mineralization was the result of reduced soil microbial biomass as well as a partial microbial substrate switch from SOC to P500, which was probably due to the toxins contained in the P500 and their utilization by actinomycetes. This study expands the knowledge of the microbial control of SOC mineralization as well as its priming effect induced by labile C to that induced by POM with a low specific surface area.