Accumulation of unfolded/misfolded proteins in endoplasmic reticulum (ER) elicits a well conserved response called the Unfolded Protein Response (UPR), which triggers the up-regulation of downstream genes involved in protein folding, vesicle trafficking, and ER-Associated Degradation (ERAD). Although the dynamic transcriptomic responses and underlying major transcriptional regulators in ER stress response in plants have been well established, the proteome changes induced by ER stress have not been reported in plants. In the current study, we found that the Arabidopsis Ler ecotype is more sensitive to ER stress than the Col ecotype. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that totally 7439 and 7035 proteins were identified from Col and Ler seedlings, with 88 and 113 differentially regulated (FC>1.3 or <0.7, P<0.05) proteins by ER stress in Col and Ler, respectively. Among them, 40 proteins were commonly up-regulated in Col and Ler, of which 10 were not up-regulated in bzip28 bzip60 double mutant (Col background) plants. Of the 19 specifically up-regulated proteins in Col comparing to that in Ler, components in ERAD, N-glycosylation, vesicle trafficking and molecular chaperones were represented. Quantitative RT-PCR showed that genes encoding 7 out of 19 proteins were not up-regulated (FC>1.3 or <0.7, P<0.05) by ER stress in both ecotypes while genes encoding 12 out of 19 proteins were up-regulated by ER stress with no obvious differences in fold change between Col and Ler. Our results experimentally demonstrated the robust ER stress response at proteome level in plants and revealed differentially regulated proteins that may contribute to differed ER stress sensitivity between Col and Ler ecotypes in Arabidopsis.