Scope Acrolein is a highly electrophilic α,β‐unsaturated aldehyde and is associated with human diseases. It is formed by Maillard reaction during food processing and could be detected in the emissions of overheated cooking oils. Consequently, humans are at risk of acrolein exposure through consumption of such prepared food. Methods and results We conducted three human studies that healthy subjects (21–30 years) were served fried foods including fried chicken and French fries from three commercial fast food restaurants. Acrolein‐related metabolites including urinary 3‐hydroxypropyl mercapturic acid (3‐HPMA), serum acrolein‐protein conjugates (Acr‐FDP), and buccal acrolein‐induced DNA damages (Acr‐dG adducts) along with GSH levels in serum or buccal cells were investigated for different times after consumption. Conclusion Urinary 3‐HPMA levels were increased after 2‐hr consumption of fried food with an elimination half‐life of 10 hr. In addition, increased Acr‐dG adducts in oral cavity were inversely correlated to buccal glutathione (GSH) levels after consumption. However, there was no significant change in systemic GSH levels or Acr‐FDP adducts in serum. These results indicate that exposure of acrolein from consuming fried food affects local oral cavity homeostasis. This may provide a possible link between intake of fried food and increased risk of upper aerodigestive tract cancers.