Despite the increasing popularity of e-cigarettes, their long-term health effects remain unknown. In animal models, exposure to e-cigarette has been reported to result in pulmonary and cardiovascular injury, and in humans, the acute use of e-cigarettes increases heart rate and blood pressure and induces endothelial dysfunction. In both animal models and humans, cardiovascular dysfunction associated with e-cigarettes has been linked to reactive aldehydes such as formaldehyde and acrolein generated in e-cigarette aerosols. These aldehydes are known products of heating and degradation of vegetable glycerin (VG) present in e-liquids. Here, we report that in mice, acute exposure to a mixture of propylene glycol:vegetable glycerin (PG:VG) or to e-cigarette-derived aerosols significantly increased the urinary excretion of acrolein and glycidol metabolites─3-hydroxypropylmercapturic acid (3HPMA) and 2,3-dihydroxypropylmercapturic acid (23HPMA)─as measured by UPLC-MS/MS. In humans, the use of e-cigarettes led to an increase in the urinary levels of 23HPMA but not 3HPMA. Acute exposure of mice to aerosols derived from PG:13C3-VG significantly increased the 13C3 enrichment of both urinary metabolites 13C3-3HPMA and 13C3-23HPMA. Our stable isotope tracing experiments provide further evidence that thermal decomposition of vegetable glycerin in the e-cigarette solvent leads to generation of acrolein and glycidol. This suggests that the adverse health effects of e-cigarettes may be attributable in part to these reactive compounds formed through the process of aerosolizing nicotine. Our findings also support the notion that 23HPMA, but not 3HPMA, may be a relatively specific biomarker of e-cigarette use.