Damage to the soil environment caused by petroleum hydrocarbon pollution has become increasingly evident. However, the chemical adsorption reaction between petroleum hydrocarbon pollutants and medium particles in unsaturated soil at actual sites remains unclear. To explore the reaction process and mechanism, a series of unsaturated experiments under sterile conditions were performed to simulate the soil state of site with n-hexadecane as a characteristic pollutant. Soil samples were collected regularly, and environmental factors, including oxidation-reduction potential (ORP) and pH, were monitored online. Through microscopic characterization and qualitative and semi-quantitative analysis methods, the morphological changes in medium particles before and after reaction were systematically analyzed. The characteristics of reaction products were determined using gas chromatography-mass spectrometry. The results showed that the consumption of oxygen (O2) and production of hexadecanoic acid [CH3(CH2)14COOH] were fundamental reasons for the decrease of ORP and pH. A hydroxyl radical (& sdot;OH) in medium particles might extract a methyl hydrogen (H) atom from n-hexadecane and turn it into an alkyl radical. The alkyl radical might eventually be oxidized by O2 in soil to 1-hexadecanol and CH3(CH2)14COOH. This study systematically explains the chemical adsorption reactions and mechanisms of petroleum hydrocarbon pollutants in unsaturated soils. In addition, it provides a theoretical basis for revealing the migration and transformation mechanisms of petroleum hydrocarbon pollutants in soil under hydrological conditions such as rainfall and water level fluctuations.