The plant hormone jasmonate regulates plant immunity and adaptive growth through orchestrating a genome-wide transcriptional program. Key regulators of jasmonate-responsive gene expression include the master transcription factor MYC2, which are repressed by the conserved Groucho/Tup1-like co-repressor TOPLESS (TPL) in the resting state. However, the mechanisms underlying TPL-mediated transcriptional repression of MYC2 activity and hormone-dependent switching between repression and de-repression remain enigmatic. Here, we report the regulation of TPL activity through reversible TPL acetylation. The histone acetyltransferase GCN5 mediates TPL acetylation, which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ (NINJA) adaptor and promotes its recruitment to MYC2 target promoters, facilitating repression. Conversely, TPL deacetylation by the histone deacetylase HDA6 weakens TPL–NINJA interaction and inhibits TPL recruitment to MYC2 target promoters, facilitating activation. In a resting state, the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis, promoting TPL repression activity. In response to jasmonate elicitation, HAD6 expression is transiently induced, decreasing TPL acetylation and repressor activity, facilitating target gene activation. Thus, the GCN5–TPL–HDA6 module maintains the homeostasis of acetylated TPL, thereby determining the transcriptional state of jasmonate-responsive genes. Our findings uncovered a mechanism by which the TPL co-repressor activity in jasmonate signaling being actively tuned in a rapid and reversible manner.