Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin.