Molybdenum disulfide (MoS2) is a potential additive for rubber enhancement. However, its poor dispersion and weak interaction with matrix limit its further application. To improve the dispersion of MoS2 in natural rubber (NR) and enhance the interaction of MoS2 with matrix, single-layered MoS2 nanosheets are prepared and further modified by cysteine. The modified MoS2 nanosheets and epoxidized natural rubber (ENR) are premixed and then added into a NR/carbon black compound to obtain a NR/carbon black/ENR-modified MoS2 nanosheets composite. The reaction between modified MoS2 nanosheets and ENR is proved by FT-IR spectra. The modified MoS2 nanosheets can improve the dispersion of carbon black in NR, and ENR can significantly improve the dispersion of modified MoS2 nanosheets in the composite. The addition of ENR-modified MoS2 nanosheets premixture can improve the abrasion resistance, increase the crosslink density and control the dynamic mechanical properties of NR. These improved properties can be attributed to the good filler dispersion and enhanced filler-matrix interaction in NR matrix.