For sustainable road construction and rehabilitation, this paper aims to evaluate the potentiality of utilizing Rubber Seed Oil (RSO) as a bio-based rejuvenator for 100 RAP recycled cold asphalt mixtures. Determination of the rejuvenation effect of RSO on the volumetric and strength characteristics of cold recycled asphalt mixtures was performed using response surface methodology (RSM). RSM was utilized to institute the optimum percentage of RSO, Total water content (TWC) and curing time (C-Time) via central composite design (CCD). Analysis of variance (ANOVA) indicated that the effect of interaction between the RSO, TWC and the C-Time was significant. For all the properties investigated, RSO seems to have more influential effect. A good agreement between the actual and predicted values was observed through the high value of the coefficient of determination (R2) and low percent error (PE) recorded. Numerical optimization suggested that the optimum contents of RSO and TWC was 0.68 and 4.7 respectively at the optimal curing duration of 15 days. © 2021 The Authors