[Objective] DNA-based stable isotope probing (DNA-SIP) is a powerful tool for tracing substrate assimilated microorganisms in complex environments. However, in recent studies involving SIP, we found that ¹³C utilization may interfere with experiment results. For example, when microcosm and DNA-SIP are applied to study straw degradation microorganism, ¹³C-labled straw is bound to be amended, but whether the amendment as well as the fertilization of soil will affect the microbial communities is still unknown.[Methods] We sampled three fertilized (Control, NPK, OM) paddy soil from Yingtan red soil ecological experimental station to study the difference of biogas emission, microbial diversity and responding species among three fertilized soils and two kinds of straw. Microcosms were conducted with ¹²C and ¹³C-labled straw amendments afterwards.[Results] We found that no difference of accumulative C emission was detected among three soils with ¹²C and ¹³C-labled straw amendments. Under oligotrophic (Control) conditions, soil bacterial communities of ¹³C-labeled treatment represented a higher diversity while microbial heterogenicity was higher in ¹²C-labeled treatment, and discrepant species were hardly detected between these two treatments. Under copiotrophic (NPK and OM) conditions, bacterial communities of ¹³C-labeled straw treatment performed no difference on both their diversity and structure, but discrepant species were detected, mainly belong to Proteobacteria and rare species. [Conclusion] Our results showed that ¹³C-labeled straw did affect bacterial communities to some extent. Hence, as labeling substrate, high abundance straw can be applied, but it need to be cautious in the following SIP experiments.