Myosin VI is the only myosin that moves toward the minus end of actin filaments, suggesting a unique biological function. Here, we show that myosin VI is present in the nucleus of mammalian cells where it colocalizes with newly transcribed mRNA and with RNA polymerase II (RNAPII) and is detected in the RNAPII complex. The colocalization and interaction of myosin VI with RNAPII require transcriptional activity. Chromatin immunoprecipitation (ChIP) demonstrates that myosin VI is recruited to the promoter and intragenic regions of active genes, encoding urokinase plasminogen activator (uPA), eukaryotic initiation factor 6 (p27/eIF6), and low-density lipoprotein receptor (LDLR), but not to noncoding, nonregulatory intergenic regions. Downregulation of myosin VI reduces steady-state mRNA levels of these genes in vivo, and antibodies to myosin VI reduce transcription in vitro. We suggest that myosin VI modulates RNAPII-dependent transcription of active genes, implicating the possibility of an actin-myosin based mechanism of transcription.