Tunnels may suffer severe damage when passing through an active fault in high-intensity earthquake zones. The present study aims to investigate the performance of an isolation layer composed of a rubber-sand mixture, an emerging trend in low-cost seismic mitigation studies. Based on the Ngong tunnel in the Nairobi-Malaba Railroad in Kenya, Africa, the effect of the rubber-sand isolation layer on the acceleration and strain of the tunnel lining was investigated through a shaking table test under small normal fault creep-slip and subsequent seismic shaking. The influences of the length of the isolation layer and the rubber content in the mixture were analyzed by numerical simulation. The results indicate that the isolation layer slightly reduces the acceleration response of the tunnel lining within the fault and obviously reduces the permanent strain of the invert and crown within the fault under small normal fault creep-slip and subsequent seismic excitation. The mitigation effect of the isolation layer is related to the length of the isolation layer and the rubber content in the mixture. In the case of this study, the length of the isolation layer is triple the fault width (influence range of the fault) and the appropriate enhancement of the rubber content of the isolation layer offers favorable conditions for mitigation effect, respectively.