Pseudomonas aeruginosa is an important opportunistic bacterial pathogen. Despite its metabolic and virulence versatility, it has not been shown to infect articular joints, which are areas that are rarely infected with bacteria in general. We hypothesized that articular joints possess antimicrobial activity that limits bacterial survival in these environments. We report that cartilages secrete a novel antimicrobial factor, henceforth referred to as the cartilage-associated antimicrobial factor (CA-AMF), with potent antimicrobial activity. Importantly, CA-AMF exhibited significantly more antimicrobial activity against P. aeruginosa strains with a functional type III secretion system (T3SS). We propose that CA-AMF represents a new class of human antimicrobial factors in innate immunity, one which has evolved to selectively target pathogenic bacteria among the beneficial and commensal microflora. The T3SS is the first example, to the best of our knowledge, of a pathogen-specific molecular target in this antimicrobial defence system.