Labile carbon (C) inputs to soil (e.g., litter and root exudation) can prime soil organic matter (SOM) decomposition, and strongly influence SOM dynamics. The direction and intensity of priming, as well as the net C balance in soil, depend on the amount and frequency of labile C inputs. Most recent priming studies are based on single C additions, which are not truly representative of common litter inputs or root exudation in terrestrial ecosystems. Here, we evaluated the effects of 14C-labeled glucose addition to soil in the same final amounts (360 μg C g−1) split into two temporal patterns: seldom (20% of microbial biomass every two months) and frequent addition (4% of microbial biomass every 10 days) on the dynamics of CO2 production and SOM priming over a 200-day incubation. For the first time, we combined enzyme kinetics with substrate-induced growth respiration and fungal diversity to monitor microbially mediated SOM mineralization in response to the labile C input frequency. Frequent glucose addition decreased 14C incorporation into microbial biomass and almost doubled cumulative priming compared to seldom addition, resulting in a net loss of SOM for seldom and frequent C additions of −94 and −367 μg C g−1 respectively. Larger priming loss of SOM with frequent C inputs was accompanied by increased activities of β-glucosidase, chitinase, and acid phosphatase, and by a shift in fungal community towards increased abundance of K-strategist fungal species (mainly Mortierellales sp. and Trichoderma sp.) capable of SOM mineralization. In conclusion, frequent labile C inputs (e.g., rhizodeposits in rhizosphere or litterfall in disturephere) to soil will stimulate a shift in fungal community structure and functions, resulting in intensive priming of SOM decomposition and CO2 losses. © 2020 Elsevier Ltd