Viroids are small, single-stranded, circular, pathogenic RNAs that do not code for proteins and thus depend on host factors for their replication and movement. They induce diseases in plants by direct interaction with host factors through a mechanism as yet unidentified. Hop stunt viroid (HSVd) has been found in a wide range of herbaceous and woody hosts. Nicotiana benthamiana is one of the most frequently used experimental systems in the study of plant–virus interactions. However, this plant is a non-host for HSVd. To go further in the study of the requirements of a plant to be a host for viroids N. benthamiana was transformed with cDNA expressing dimeric (+) HSVd. Correct processing to the circular (+) monomers was always observed, demonstrating that N. benthamiana has the appropriate machinery to cleave and circularize (+) HSVd molecules. Additionally, N. benthamiana plants agroinoculated with the dimeric (–) HSVd accumulated the circular and linear (+) monomers, indicating that (–) HSVd transcripts can be used as template for the RNA–RNA transcription. Grafting assays showed that HSVd is able to move to distal plant parts. In addition, alterations in the normal flower development were observed. Taken together, these results indicate that deficiencies in the interaction with host factors related either to the early steps of the replication process or to cell to cell movement appear to be the factors that limit infectivity of HSVd in N. benthamiana. These transgenic plants can be an useful tool to study the HSVd–host factor interactions.