Bio-oils are increasingly used to enhance petroleum bitumen’s intermediate and low-temperature characteristics regarding their economic and environmental benefits. Research has shown a beneficial impact of nanomaterials on altering bitumen’s high-temperature characteristics. The present study uses a mixture of rice bran oil and nano-calcium oxide to improve the performance of bitumen in all three temperature conditions. Rice bran oil was added to pure bitumen at doses of 3, 5, and 7 % relative to the weight of bitumen. The rheological tests (dynamic shear rheometer, multiple stress creep recovery, linear amplitude sweep, and bending beam rheometer) demonstrated that using rice bran oil in bitumen enhanced its performance at intermediate and low-temperatures but declined its performance at high-temperatures. Therefore, to improve the performance at high-temperatures, the bituminous sample containing 5 % rice bran oil was combined with 1, 3, and 5 % nano-calcium oxide, followed by repeating the rheological tests on them. The samples modified with nano-calcium oxide showed increased friction and shear stress, viscosity, and rutting parameters. Besides, the samples exhibited reduced sensitivity to aging compared to the bio-oil samples. However, the non-recoverable creep compliance and percentage of recovery showed improvements. The fatigue life of bio-oil samples decreased after adding nano-calcium oxide. However, the fatigue life remained higher than the base bitumen sample when adding 1 % nano-calcium oxide. Fourier-transform infrared spectroscopy analysis showed that a new chemical reaction occurred due to adding nano-calcium oxide to bitumen containing rice bran oil. Besides, the scanning electron microscope image revealed that the nano-calcium oxide particles are uniformly and homogeneously distributed within the bitumen sample containing 5 % rice bran oil. Consequently, the mix containing 5 % rice bran oil and 3 % nano-calcium oxide increases the resistance against high-temperature failures and improves the low-temperature performance compared to the base bitumen sample.