The synthesis and characterization of tantalum oxide (Ta2O5) nanoparticles (NPs) as new X-ray contrast media for microcomputed tomography (μCT) imaging of articular cartilage are reported. NPs, approximately 5–10 nm in size, and possessing distinct surface charges, were synthesized using phosphonate (neutral), ammonium (cationic), and carboxylate (anionic) ligands as end functional groups. Assessment of a cartilage defect in a human cadaver distal metacarpophalangeal (MCP) joint with the ammonium nanoparticles showed good visualization of damage and preferential uptake in areas surrounding the defect. Finally, an optimized nontoxic cationic NP contrast agent was evaluated in an in vivo murine model and the cartilage was imaged. These nanoparticles represent a new type of contrast agent for imaging articular cartilage, and the results demonstrate the importance of surface charge in the design of nanoparticulate agents for targeting the surface or interior zones of articular cartilage.