DNA-dependent RNA polymerases catalyse the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. POLR3D is a specific peripheral component of RNA polymerase III (Pol III), which synthesises small non-coding RNAs including 5S rRNA, snRNAs, tRNAs and miRNAs from at least 500 distinct genomic loci. POLR3D assembles with POLR3E (RPC5) to form a subcomplex that binds the Pol III core.

Within the transcription cycle, the POLR3D protein enables recruitment of Pol III at the transcription initiation site and drives initiation from both type 2 and type 3 DNA promoters. It is also required for efficient transcription termination and reinitiation. The POLR3D gene belongs to the HGNC gene group of RNA polymerase subunits, and the protein is a member of the eukaryotic RPC4/POLR3D RNA polymerase subunit family. It is located in the nucleus.

Beyond transcription, Pol III plays a key role in sensing and limiting infection by intracellular bacteria and DNA viruses. It acts as a nuclear and cytosolic DNA sensor in the innate immune response, and can sense non-self double-stranded DNA that serves as a template for transcription into double-stranded RNA. Non-self Pol III transcripts, such as Epstein-Barr virus-encoded RNAs (EBERs), induce type I interferon and NF-kappa-B through the RIG-I pathway. The gene was named for its ability to complement a temperature-sensitive mutant from the BHK-21 Syrian hamster cell line, which blocks progression through the G1 phase of the cell cycle at nonpermissive temperatures; hence the earlier name BN51 (BHK21) temperature sensitivity complementing.