Pan-BD2 inhibitors have been shown to retain an antileukemia effect and display less dose-limiting toxicities than pan-BET inhibitors. However, it is necessary to consider the potential off-target toxicity associated with the inhibition of four BET BD2 proteins. To date, no BRD4 BD2 domain selective inhibitor has been reported. Based on our previous pan-BD2 inhibitor 12 (XY153), we successfully identified 16o (XY221) as the first BRD4 BD2-selective inhibitor. 16o demonstrated potent binding affinity for BRD4 BD2 (IC50 = 5.8 nM), along with high pan-BD2 selectivity (667-fold over BRD4 BD1) and BRD4 BD2 domain selectivity (9-32-fold over BRD2/3/T BD2). The BRD4 BD2 selectivity of 16o was further confirmed by the BLI assay, showing 66-144-fold selectivity over other BET BD2 domains. 16o exhibited good liver microsomal stability (T1/2 > 120 min) and pharmacokinetic properties (F = 13.1%). These data indicate that 16o may serve as a valuable candidate for BRD4 BD2 advancing epigenetic research.