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BRI1-EMS-SUPPRESSOR 1 (BES1) transcription factor is closely associated with the brassinosteroid (BR) signaling pathway and plays an important role in plant growth and development. SLB3 is a member of BES1 transcription factor family and its expression was previously shown to increase significantly in tomato seedlings under drought stress. In the present study,we used virus-induced gene silencing (VIGS) technology to downregulate SLB3 expression to reveal the function of the SLB3 gene under drought stress further. The downregulated expression of SLB3 weakened the drought tolerance of the plants appeared earlier wilting and higher accumulation of H2O2 and O2–·, decreased superoxide dismutase (SOD) activity, and increased proline (PRO) and malondialdehyde (MDA) contents and peroxidase (POD) activity. Quantitative real-time PCR (qRT-PCR) analysis of BR-related genes revealed that the expression of SlCPD, SlDWARF and BIN2-related genes was significantly upregulated in SLB3-silenced seedlings under drought stress, but that the expression of TCH4-related genes was downregulated. These results showed that silencing the SLB3 gene reduced the drought resistance of tomato plants and had an impact on the BR signaling transduction which may be probably responsible for the variation in drought resistance of the tomato plants.