Abstract: To assess the toxic effects of crotonaldehyde on human bronchial epithelial cells BEAS-2B, cell counting kit-8(CCK-8) assay was adopted to detect the viability of BEAS-2B cells; immunofluorescence assay to the accumulation and location of autophagosomes; and Western Blot assay to autophagy-marker microtubule-associated protein 1 light chain 3 beta (LC3B-Ⅱ), the variations of two pathways of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK)/Unc-51-like kinase 1 (ULK1). The results indicated that:1) The viability of BEAS-2B cells decreased significantly after exposure to crotonaldehyde. 2) Crotonaldehyde significantly promoted the accumulation of autophagosomes and changed the expression level of LC3B-Ⅱ. Induced autophagy had apparent time-effect relationship. The level of autophagy in BEAS-2B cells was up-regulated prior to down-regulation with the increase of exposure time. 3) The relative expression of phospho-(P-) PI3K, P-Akt and P-4E-binding protein1 (4E-BP1) in PI3K/Akt/mTOR pathway decreased, while that of P-mTOR and P-ribosomal protein S6 kinase (p70 S6K) decreased first and then increased. The activities of LKB1, AMPK, and ULK1 in LKB1/AMPK/ULK1 pathway increased. Collectively, the two pathways mediated the up-regulation of autophagy in BEAS-2B cells exposed to crotonaldehyde.