Argonaute proteins are the effectors of small RNA-dependent gene-silencing pathways. In the cytoplasm, they are incorporated into large mobile ribonucleoprotein (RNP) complexes that travel along microtubules. We used a genetic screen to identify the microtubule-associated motor that interacts with Ago1-containing RNPs. Here, we report that activity of the kinesin family member Cut7 is important for biogenesis and/or stability of Ago1-containing RNPs in the cytoplasm. Results from pulldown and coimmunoprecipitation assays indicate that Cut7 interacts with Ago1 as well as its two cognate binding proteins, Dcr1 and Rdp1. Loss of Cut7 activity was associated with increased levels of reverse centromeric transcripts, presumably because of a defect in post-transcriptional gene silencing. Overexpression of the Ago1-binding region of Cut7 resulted in loss of microscopic Ago1-containing RNPs. Together, these results suggest that microtubule motor proteins function in the biogenesis and function of gene-silencing machinery in the cytoplasm.