Clostridium difficile is associated with a spectrum of clinical manifestations ranging from asymptomatic carriage to severe life-threatening pseudomembranous colitis. Current perspectives indicate that C difficile pathogenesis is a multifactorial disease process dictated by pathogenic toxin production, gut microbial dysbiosis, and altered host inflammatory responses. This article summarizes recent findings underpinning the cellular and molecular mechanisms regulating bacterial virulence and sheds new light on the critical roles of the host immune response, intestinal microbiota, and metabolome in mediating disease pathogenesis.