Over the years, a reduced upper limit on sulfur content in marine-vehicle fuel has created a situation where a major diversion of sulfur into bitumen will be noticeable in the near future. In addition to the naturally occurring sulfur compounds in bitumen, sulfur is known as a promising admixture for the bitumen matrix. Despite the hydrophobic nature of elemental sulfur, which is commercially used as a modifier or extender in asphalt mixtures, the moisture susceptibility of sulfurized bitumen is one of the concerns challenging the use of sulfur in bitumen. In this study, we used laboratory experiments to examine how the effect of moisture conditioning varies between low-sulfur bitumen and high–sulfur bitumen. We also used molecular modeling in a framework of density functional theory (DFT) to gain insights into the molecular mechanisms by which a hydrophobic substance (elemental sulfur) acts as a hydrophile to attract water molecules and increase the moisture susceptibility of bitumen. The results show that high–sulfur bitumen has significantly more strain accumulation and shear thinning when exposed to water for an extended period. Based on our molecular-level analysis geared toward a DFT approach, the affinity of polymeric chains of sulfur to water could be a driving force for the moisture susceptibility of sulfurized bitumen. The presence of polymeric chains of sulfur, even at ambient temperature, can be attributed to that portion of polysulfides formed at high temperatures that remain in the matrix after dropping the temperature. Another mechanism proposed is the thermal decomposition of polysulfides upon heating, leading to the formation of thiols (RS-H), which can easily interact with water molecules. Considering the higher concentration of Sdouble bondO bonds in sulfurized bitumen, a part of the moisture susceptibility could also be attributed to those sulfur compounds that have been functionalized with hydrophilic groups, such as sulfonate, sulfoxide, or other functional groups containing oxygen. The high concentrations of sulfoxide group (Sdouble bondO), particularly in aged bitumen, and their ability to set up H-bonding interactions, make them potential candidates for effective interactions with water molecules.