Low molecular weight polyisoprene (LIR) with different microstructure were prepared by anionic polymerization and applied to natural rubber (NR) reinforced with carbon black. In order to explore the effect of polymerization reaction temperature and tetrahydrofuran (THF) on the microstructure of polyisoprene. The results of FT-IR and 1H-NMR show that the lower the reaction temperature and the more the THF content, the higher the content of 3,4-structure of polyisoprene. In particularly, THF has a significant effect on the microstructure of polyisoprene. The DSC and TGA analysis show that the glass transition temperature and thermal decomposition temperature of LIR increase with the increase of the 3,4 structure content of LIR. The LIR with different microstructure was applied to the formulation of natural rubber (NR) reinforced with carbon black. The influence of the microstructure of LIR on the properties of vulcanized rubber was compared. The results show that, compared with aromatic oils, LIR can improve the tensile properties and dynamic mechanical properties of vulcanizates, and LIR with high 3, 4-structure is beneficial to improve the wet slip resistance of composites and LIR with high 1, 4-structure is beneficial to improve the rolling resistance of composites.