The aim of this study was to examine the ultrastructure and cytoskeletal organization in human normal and Osteoarhritic (OA) chondrocytes, exposed to interleukin-1β (IL-1β) and cyclic hydrostatic pressure (HP). Morphological examination by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed differences between normal and OA chondrocytes at the nuclear and cytoplasmic level. IL-1β (5 ng/mL) induced a decrease of the number of mitochondria and Golgi bodies and a significant increase on the percentage of cells rich in vacuolization and in marginated chromatin. Cyclical HP (1–5 MPa, 0.25 Hz, for 3 h) did not change the morphology of normal chondrocytes, but had a beneficial effect on OA chondrocytes increasing the number of organelles. Normal and OA cells subjected to IL-1β and HP recovered cytoplasmic ultrastructure. Immunofluorescence (IF) examination of normal chondrocytes showed an actin signal polarized on the apical sides of the cytoplasm, tubulin and vimentin uniformly distributed throughout cytoplasm and vinculin revealed a punctuated pattern under the plasma membrane. In OA chondrocytes, these proteins partially lost their organization. Stimulation with IL-1β caused, in both type of cells, modification in the cytoskeletal organization; HP counteracted the negative effects of IL-1β. Our results showed structural differences at nuclear, cytoplasmic and cytoskeletal level between normal and OA chondrocytes. IL-1β induced ultrastructural and cytoskeletal modifications, counteracted by a cyclical low HP.