In this study, the protein expression profiles of zebrafish embryos under chlorpyrifos (CPF) stress were investigated. Zebrafish embryos were exposed to 0.25 mg/L CPF, and embryo samples were collected until 24 h post-fertilization (hpf). To gain a better understanding of the response of zebrafish embryos to CPF exposure, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with mass spectrometry was employed to carry out a comparative proteomic analysis. Total proteins were extracted from the control and treated samples, separated by 2D PAGE, and visualized by silver staining. A total of 59 protein spots showed reproducible changes compared with the control. Of these 59 spots, 19 were selected and subjected to matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight mass spectrometry (TOF/TOF) analysis; 9 differentially expressed proteins were successfully identified, including 3 up-regulated proteins and 6 down-regulated proteins. The increased expression of 3 proteins associated with detoxification and stress response suggested that the activation of protective proteins was required in zebrafish embryos exposed to CPF. On the other hand, the decreased expression of 6 proteins is mainly involved in cytoskeleton structure, protein translation, signal transduction and lipoprotein metabolism. These data may help us understand the functions and the molecular mechanisms of these proteins in zebrafish embryos' response to CPF exposure.