Castor oil (CO) functionalized by isocyanate groups (-NCO) is proposed as a novel bio-based reactive polyurethane (PU) for bitumen modification. This work presents a comparative analysis conducted on blends of bitumen and 2 wt.% of a PU prepolymer prepared by NCO-functionalization of castor oil. Four preparation procedures were evaluated, which resulted from the combination of two processing times (1h or 24h, at 90ºC) followed by two different post-treatments (water addition or ambient curing for up to 6 months). It was found that the degree of modification attained after posttreatment depends on the previous processing conditions. Thus, short processing times are required if the binder is further subjected to ambient curing. Instead, the success of the water-addition modification falls on a previous long processing step. As revealed by rheological tests, ambient curing was seen to be by far a more efficient way of modification if compared to direct addition of water, and makes clear that the resulting binder evolves towards a better performance when in service. In that sense, Thin Layer Chromatography tests, Modulated DSC and AFM images demonstrated a more complex microstructure characterized by the presence of a larger content of molecules with higher polarity, size and molecular weight.