This study investigates the temperature-dependent frictional behavior of MoS2 in humid environments within the context of a long-standing debate over increased friction due to oxidation processes or molecular adsorption. By combining sliding friction experiments and density functional theory (DFT)-based first-principles simulations, it aims to clarify the role of water molecule adsorption in influencing frictional properties of MoS2, addressing the challenge of identifying interfacial bonding behavior responsible for friction in such conditions. Sliding experiments revealed that magnetron-sputtered MoS2 exhibits a reduction in the coefficient of friction (COF) with an increase in temperature from 25 to 100 °C under 20 and 40% relative humidity. This change in the COF obeys the Arrhenius law, presenting an energy barrier of 0.165 eV, indicative of the temperature-dependent nature of these frictional changes and suggests a consistent frictional mechanism. DFT simulations showed that H2O molecules are adsorbed at MoS2 vacancy defects with adsorption energies ranging from −0.56 to −0.17 eV, which align with the experimentally determined energy barrier. Adsorptive interactions, particularly the formation of stable H···S interfacial hydrogen bonds at defect sites, increase the interlayer adhesion and impede layer shearing. TEM analysis confirms that although MoS2 layers align parallel to the sliding direction in humid conditions, the COF remains at 0.12, as opposed to approximately 0.02 in dry air. This demonstrates that parallel layer alignment does not notably decrease the COF, underscoring humidity’s significant role in MoS2’s COF values, a result also supported by the Arrhenius analysis. The reversibility of the physisorption process, demonstrated by the recovery of the COF in high-temperature humid environments, suggests its dynamic nature. This study yields fundamental insights into MoS2 interfaces for environments with variable humidity and temperature, crucial for demanding tribological applications.