Wear particles are phagocytosed by macrophages, resulting in cellular activation and the release of pro-inflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty (TJA) failure. During this pathological process, tumor necrosis factor (TNF)-α plays an important role in wear particle-induced osteolysis. Therefore, in this study, we used adenovirus-mediated small interfering RNA (siRNA) targeting TNF-α to suppress the TNF-α release from activated macrophages in response to titanium particles. Our results showed that recombinant adenovirus (Ad-TNF-α-siRNA) suppressed the TNF-α release from activated macrophages in response to titanium particles, and reduced titanium particle-induced osteoclastogenesis and bone resorption in the presence of receptor activator of nuclear factor-κB ligand (RANKL). In addition, the conditioned medium of macrophages challenged with titanium particles (Ti CM) stimulated osteoprogenitor RANKL expression. The conditioned medium of macrophages challenged with titanium particles and Ad-TNF-α-siRNA (Ti-Ad CM) reduced the mRNA expression in MC3T3-E1 cells compared to Ti CM. Based on these data, TNF-α strongly synergizes with RANKL to promote osteoclast differentiation. Furthermore, TNF-α promoted osteoclast differentiation by stimulating osteoprogenitor RANKL expression. Ad-TNF-α-siRNA effectively suppressed osteoclast differentiation and bone resorption following exposure to titanium particles in the presence of RANKL. In addition, recombinant adenovirus (Ad-TNF-α-siRNA) does not have a toxic effect on the murine macrophage cell line, RAW264.7. Consequently, it can be concluded that recombinant adenovirus-mediated siRNA targeting TNF-α (Ad-TNF-α-siRNA) may provide a novel therapeutic approach for the treatment of periprosthetic osteolysis.