A novel active vibration isolation system using negative stiffness structure (active system with NSS) for low excitation frequency ranges (< 5 Hz) is developed successfully. Here, the negative stiffness structure (NSS) is used to minimize the attraction of vibration. Then, the fuzzy sliding mode controller (FSMC) is designed to improve the vibration isolation performance of the active system with NSS. Based on Lyapunov stability theorem, the fuzzy control rules are constructed. Next, the experimental apparatus is built for evaluating the isolation efficiency of the proposed system controlled by the FSMC corresponding to various excitation conditions. In addition, the isolation performance of the active system with NSS, the active system without NSS and the passive the system with NSS is compared. The experimental results confirmed that the active system with NSS gives better isolation efficiency than the active system without NSS and the passive system with NSS in low excitation frequency areas.