No-tillage continuously contribute organic matter and increase carbon sequestration in the soil. However, global warming may stimulate microbial activity and accelerate soil organic matter (SOM) decomposition, also especially in the topsoil. Understanding the responses of microbial utilization of SOM accumulated under no-tillage to higher temperatures is necessary to develop management strategies to sustain soil fertility. Determine the response of microbial extracellular enzyme activities and SOM decomposition to long-term warming by incorporation of 14C-labelled maize litter to in situ warmed soil from no-tillage and till systems. We compared decomposability of litter and SOM in soils from a 5-year in situ experiment with two tillage systems (Till and No-till) and under two temperature levels: ambient temperature and continuous soil warming (+1.6 °C at 5 cm soil depth). We hypothesized that decomposition of crop litter (14C-labeled maize) at increasing temperature (15, 21, and 27 °C for 59 days) will be more intensive in warmed soil vs. ambient and the effects should be stronger under No-till vs. Till system. As expected, soil from field warming had always higher total CO2 efflux (from 5.5 to 12%) than the non-warmed counterpart in Till and No-till systems. Five-year warming increased temperature sensitivity (Q10) of CO2 efflux for 2-folds under No-tillage vs. Till. Litter decomposition (measured as 14CO2) in No-till with warming was 7.9% greater than in No-tillage without warming. Three extracellular enzymes (β-glucosidase, chitinase, and sulfatase) had higher activities under warming in No-till but not under Till system. Our results demonstrated that the 5-year in situ warming accelerated preferential microbial utilization of labile SOM fractions in No-till notably, and from stable SOM in Till system. Consequently, warming will accelerate SOM decomposition in future and this acceleration will be especially pronounced on the labile SOM under No-tillage systems.