Abstract Brassinosteroids (BRs) are a group of steroidal hormones involved in plant development. Although the BR biosynthesis pathways are well characterized, the BR inactivation process, which contributes to BR homeostasis, is less understood. Here, we show that a member of the BAHD (for benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, and deacetylvindoline 4-O-acetyltransferase) acyltransferase family may play a role in BR homeostasis in Arabidopsis (Arabidopsis thaliana). We isolated two gain-of-function mutants, brassinosteroid inactivator1-1Dominant (bia1-1D) and bia1-2D, in which a novel BAHD acyltransferase-like protein was transcriptionally activated. Both mutants exhibited dwarfism, reduced male fertility, and deetiolation in darkness, which are typical phenotypes of plants defective in BR biosynthesis. Exogenous BR treatment rescued the phenotypes of the bia1-1D mutant. Endogenous levels of BRs were reduced in the bia1-1D mutant, demonstrating that BIA1 regulates endogenous BR levels. When grown in darkness, the bia1 loss-of-function mutant showed a longer hypocotyl phenotype and was more responsive to exogenous BR treatment than the wild-type plant. BIA1 expression was predominantly observed in the root, where low levels of BRs were detected. These results indicate that the BAHD acyltransferase family member encoded by BIA1 plays a role in controlling BR levels, particularly in the root and hypocotyl in darkness. Taken together, our study provides new insights into a mechanism that maintains BR homeostasis in Arabidopsis, likely via acyl conjugation of BRs.