The auxin IAA is a vital plant hormone in controlling growth and development, but our knowledge about its complicated biosynthetic pathways and molecular regulation are still limited and fragmentary. cytokinin induced root waving 2 (ckrw2) was isolated as one of the auxin-deficient mutants in a large-scale forward genetic screen aiming to find more genes functioning in auxin homeostasis and/or its regulation. Here we show that CKRW2 is identical to Histone Monoubiquitination 1 (HUB1), a gene encoding an E3 ligase required for histone H2B monoubiquitination (H2Bub1) in Arabidopsis. In addition to pleiotropic defects in growth and development, loss of CKRW2/HUB1 function also led to typical auxin-deficient phenotypes in roots, which was associated with significantly lower expression levels of several functional auxin synthetic genes, namely TRP2/TSB1, WEI7/ASB1, YUC7 and AMI1. Corresponding defects in H2Bub1 were detected in the coding regions of these genes by chromatin immunoprecipitation (ChIP) analysis, indicating the involvement of H2Bub1 in regulating auxin biosynthesis. Importantly, application of exogenous cytokinin (CK) could stimulate CKRW2/HUB1 expression, providing an epigenetic avenue for CK to regulate the auxin homeostasis. Our results reveal a previously unknown mechanism for regulating auxin biosynthesis via HUB1/2-mediated H2Bub1 at the chromatin level.