Aim To study the mechanism of benzo(a)pyrene (BaP) affecting autophagy in human umbilical vein endothelial cell (HUVEC). Methods HUVECs were treated with BaP (2.5,5, 10 μmol/L) for 24 h. The degradation of autophagosome and its contents, the expressions of target proteins microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (p62), Beclin-1, autophagy-related protein 5 (Atg5), Atg7, Atg12, cathepsin B (CTSB), cathepsin D (CTSD), syntaxin 17 (STX17), lysosomal associated membrane protein 2 (LAMP2), the number and function of lysosomes, and the phosphorylation levels of related upstream key regulatory proteins serine-threonine protein kinase (Akt), extracellular regulated protein kinase (ERK) and transcription factor EB (TFEB) were detected respectively by indirect immunofluorescence, Western blot, acridine orange staining and monodansyl cadaverine staining.Results In HUVEC of BaP exposure group, the test results showed that:(1)The level of LC3 puncta and the ratio of LC3Ⅱ/LC3Ⅰ increased, the expressions of key autophagy initiation proteins (Beclin-1, Atg5, Atg7 and Atg12) increased, and the phosphorylation of Akt protein decreased significantly; (2)The levels of p62 puncta and p62 protein increased significantly; (3)The number of lysosomes increased, accompanied by the increased expressions of lysosomal characteristic hydrolases (CTSB, CTSD), and the phosphorylation levels of ERK and TFEB increased accordingly; (4)The key proteins STX17 and LAMP2 regulating the fusion of autophagy and lysosome decreased. Conclusion BaP inhibits the normal autophagic flux of HUVEC by reducing the expression levels of STX17 and LAMP2.