Biochar has been widely proposed to be valuable in the sequestering of carbon (C) in soil due to its chemical and biological recalcitrance. However, whether biochar could cause soil positive priming effects (PEs), which offset the effects of soil organic C sequestration, has raised a very controversial issue and debate recently. Changed soil properties, like microbial community composition, caused by biochar addition, might induce different primed CO2 following substrate addition, compared to soil which never received biochar. However, this remains largely unknown. This study aimed to understand the substrate-induced PEs in biochar-amended soil and the microbial mechanisms involved. Using 13C analysis, a further 28 days of laboratory incubation was conducted after incorporation of biochar for 431 days of pre-incubation to investigate primed soil CO2 emissions induced by the addition of sucrose and Miscanthus giganteus (Miscanthus), in both biochar free soil (L1) and biochar (produced at 350 and 700 °C)-amended soils (L2 and L3). Biochar-amended soils had larger substrate-induced PEs. Larger primed soil C losses (311 μg CO2-C g−1 soil) were observed following Miscanthus feedstock addition in BC700-amended soil (L3 + Miscanthus), compared to soil without BC700 (193 μg CO2-C g−1 soil) (L3). The changes in soil microbial community composition, indicated by PCA analysis of PLFAs, especially actinomycetes and Gramme-negative bacteria, might be responsible for the larger substrate (Miscanthus and sucrose)-induced PEs observed in biochar-amended soils after 431 days compared to biochar-free soils.