AtDjB1 is a member of the Arabidopsis thaliana J-protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1-1) exhibited greater ABA sensitivity compared with the wild-type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA-responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1-1 plants exhibited higher glucose levels and greater glucose sensitivity in the post-germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1-1 than WT seedlings. Taken together, higher glucose levels in atj1-1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.