ABSTRACT Secondary cell wall (SCW) synthesis is a critical process of wood formation. Extensive studies have identified NAC and MYB transcription factors as key regulators of SCW thickening. Currently, no studies reveal the involvement of BTB/POZ proteins during wood formation. In this study, we investigated the role of a BTB/POZ protein during wood formation in Populus alba × Populus glandulosa. PagBTB74 overexpression ( PagBTB74‐OE ) and dominant repression ( PagBTB74‐SRDX ) transgenic plants exhibited narrowed xylem and thinner SCWs of xylem cells. Conversely, RNAi silencing ( PagBTB74‐RNAi ) transgenic plants displayed opposite phenotypes with widened xylem and thickened walls. Integrated chromatin immunoprecipitation (ChIP), RNA‐seq and yeast one‐hybridisation (Y1H) analyses identified PagVDN6‐C1 , PagCesA7 and PagXTH4 as direct targets of PagBTB74. RNA‐seq and effector‐reporter assays showed that these three genes were downregulated by PagBTB74, consisting with the result that PagBTB74 had transcriptional repression activities. Our study represents the first report of a BTB transcription factor as a negative regulator of SCW biosynthesis during wood formation, and this regulation may occur through direct repression of the key genes of SCW biosynthesis, including PagVND6‐C1 , PagCesA7 and PagXTH4 .