Cigarette smoking is one of the main sources of toxic chemical exposure to humans and is the greatest cause of progression of vascular disease. Crotonaldehyde is one of the major constituents of cigarette smoke and a product of endogenous lipid peroxidation. Thioredoxin reductase (TrxR) is a key element of the intact thioredoxin (Trx) system, which is predominant in modulating the intracellular redox homeostasis. In this study, we showed the effects of crotonaldehyde on the induction of TrxR1 expression in human endothelial cells. Crotonaldehyde exposure caused notably increased phosphorylation of p38 mitogen-activated protein kinase (MAPK). Introduction of siRNA against nuclear factor erythroid 2-related factor 2 (Nrf2) downregulated TrxR1 expression. Furthermore, treatment with auranofin, a TrxR inhibitor, significantly increased the death rate of crotonaldehyde-exposed cells. In summary, these results suggest a role for TrxR1 upregulation through p38 MAPK-Nrf2 activation in the adaptive response of human endothelial cells to crotonaldehyde.