--- - |2- Abstract Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin‐8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F‐actin with cytochalasin‐D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide‐treated cells. Examination of the individual roles of vimentin and cytokeratin‐8 IFs in the existence of vacuoles was accomplished using small interfering RNA–mediated RNA interference to knock down either vimentin or cytokeratin‐8 expression. Reduction of cytokeratin‐8 expression was associated with a less‐vacuolated cell morphology. These data demonstrate that cytokeratin‐8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration. - Journal of Cellular Physiology, EarlyView.