In this study, we characterize the changes in nucleolar morphology and its dynamics induced by the recently introduced compound CX-5461, an inhibitor of ribosome synthesis. Time-lapse imaging, immunofluorescence and ultrastructural analysis revealed that exposure of cells to CX-5461 has a profound impact on their nucleolar morphology and function: nucleoli acquired a compact, spherical shape and display enlarged, ring-like masses of perinucleolar condensed chromatin. Tunnels consisting of chromatin developed as transient structures running through nucleoli. Nucleolar components involved in rRNA transcription, fibrillar centres and dense fibrillar component with their major constituents ribosomal DNA, RNA polymerase I and fibrillarin maintain their topological arrangement but become reduced in number and move towards the nucleolar periphery. Nucleolar changes are paralleled by an increased amount of the DNA damage response indicator γH2AX and DNA unwinding enzyme topoisomerase I in nucleoli and the perinucleolar area suggesting that CX-5461 induces torsional stress and DNA damage in rDNA. This is corroborated by the irreversibility of the observed altered nucleolar phenotypes. We demonstrate that incubation with CX-5461, apart from leading to specific morphological alterations, increases senescence and decreases cell replication. We discuss that these alterations differ from those observed with other drugs interfering with nucleolar functions.