Hydrogen sulfide (H2S) is a hazardous and corrosive byproduct generated during heavy oil recovery, particularly during hot water flooding. Previous studies on factors influencing H2S generation during hot water flooding have been mainly focused on high temperatures (>250 °C), which may not accurately represent reservoir conditions. Moreover, the concentration of H2S produced by hot water flooding at low temperatures exceeds the standard. In this study, experiments were conducted on hot water flooding at low temperatures (110–150 °C). The mechanism of reactants and reaction conditions on H2S generation was investigated. The results showed that thermochemical sulfate reduction (TSR) was the primary reaction type responsible for H2S generation, while aquathermolysis and pyrite oxidation reactions weakly or did not occur. The reactivity of TSR was directly proportional to reaction temperature and time, while inversely proportional to reaction pH. The formation of oxidants ([MgSO4]CIP and HSO4–) was also found to be crucial for TSR initiation. Unstable organic sulfur-containing compounds were oxidized to produce CO2, H2S, SO3, and solid bitumen, which further sustained the autocatalytic reaction. Low temperature TSR was found to consume the saturated fraction in heavy oil and convert inorganic sulfur to organic sulfide. The increase in pH inhibited the conversion of inorganic sulfur to organic sulfur, resulting in a higher percentage of the saturated fraction. This study provides new insights into the low temperature TSR reaction mechanism and the origin of H2S, which can aid in better understanding and mitigation of the associated risks during heavy oil recovery.