Abstract SMYD3 methyltransferase is nearly undetectable in normal human tissues but highly expressed in several cancers, including breast cancer, although its contributions to pathogenesis in this setting are unclear. Here we report that histone H2A.Z.1 is a substrate of SMYD3 that supports malignancy. SMYD3-mediated dimethylation of H2A.Z.1 at lysine 101 (H2A.Z.1K101me2) increased stability by preventing binding to the removal chaperone ANP32E and facilitating its interaction with histone H3. Moreover, a microarray analysis identified cyclin A1 as a target coregulated by SMYD3 and H2A.Z.1K101me2. The colocalization of SMYD3 and H2A.Z.1K101me2 at the promoter of cyclin A1 activated its expression and G1–S progression. Enforced expression of cyclin A1 in cells containing mutant H2A.Z.1 rescued tumor formation in a mouse model. Our findings suggest that SMYD3-mediated H2A.Z.1K101 dimethylation activates cyclin A1 expression and contributes to driving the proliferation of breast cancer cells. Cancer Res; 76(20); 6043–53. ©2016 AACR.