Pol III functions in the nucleus for transcription and cytoplasm as a viral-DNA sensor, but how cells maintain the cytoplasmic-to-nuclear partitioning of Pol III activity is unknown. Here, we found that Pol III forms transcription-dependent protein clusters in the nucleus and preferentially associates with assembly factors in the cytoplasm. Acute protein degradation of Pol III subunits followed by transient protein labeling showed that nuclear and cytoplasmic Pol III response differently after the loss of their subunits.