DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G₁–S-phase transition, is phosphorylated on Thr²⁸⁶ by GSK3β (glycogen synthase kinase 3β) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr²⁸⁸ rather than Thr²⁸⁶. Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr²⁸⁶, not Thr²⁸⁸, in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr²⁸⁶ phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3β inhibitors or GSK3β RNAi. DYRK1B expression causes a G₁-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21^CIP1 (21 kDa CDK-interacting protein 1) and p27^KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr²⁸⁶-CCND1 kinase that acts independently of GSK3β to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.