Polyethylene (PE) wear particles are associated with the osteolysis seen in aseptic loosening that leads to orthopaedic implant failure. While cells of the monocyte/macrophage lineage are implicated, evidence is now emerging that osteoblastic cells may also be affected by PE. In this study we investigated the effect of PE particles on osteoblasts, using a novel in vitro cell culture system that was developed to juxtapose cells and PE particles, replicating the 3-dimensional (3D) environment near implants. This system allowed normal human bone-derived cells (NHBC) to undergo differentiation into a mature osteocyte-like phenotype over a 21-28-day culture period. PE particles induced an increase in mRNA expression of the osteocyte markers E11, DMP-1 and SOST/sclerostin. NHBC responded to PE particles by increasing the mRNA expression of several genes associated with osteoclast formation and activity (RANKL, IL-8 and M-CSF) and decreased the expression of the osteoclast antagonist, OPG. PE also appeared to induce a switch in the RUNX2 control of gene expression from that of promoting matrix production (type I collagen) to inducing the expression of pro-osteoclastogenic genes. These results suggest that PE particles switch mature osteoblastic cells from an anabolic to a more catabolic phenotype. This concept was further supported by the finding that PE-induced expression of RANKL mRNA in the mouse osteocyte cell line, MLO-Y4. Overall, our results suggest that PE particles directly induce a change in the phenotype of mature osteoblasts and osteocytes, consistent with the net loss of bone near orthopaedic implants.