Trans-1,4-poly(butadiene-co-isoprene) copolymer rubber (TBIR) and wet mixing natural rubber master. batch (W-NR) were used to modify natural rubber (NR) nanocomposites with different polymer network structures and filler network structures. The structure and properties of NR, NR/W-NR, NR/TBIR and NR/W-NR/TBIR rubber nanocomposites were investigated by means of carbon black packing dispersion apparatus, differential scanning calorimetry and equilibrium swelling method, etc. Compared with NR and NR/W-NR vulcanizates, the addition of TBIR component constructed special polymer network structures including sulfide bonds and polymer crystals acting as physical crosslinks in the NR matrix, which improved the modulus and green strength of the NR matrix, provided better filler dispersion, and improved filler-rubber interactions. Therefore, compared with NR and NR/W-NR vulcanizates, the NR/TBIR(90/10) and NR/W-NR/TBIR(40/66.5/10) vulcanizates showed improved DIN abrasion resistance, enhanced tensile fatigue property, reduced rolling resistance, and better aging resistance. The NR/W-NR/TBIR(40/66.5/10) vulcanizate prepared by combining the advantages of W-NR and TBIR together presented much lower rolling resistance, as well as excellent comprehensive properties. This work provides a facial and effective regulation way to adjust the polymer network structure and filler network structure, and fabricate NR nanocomposites with excellent comprehensive properties.