Background. Health care-associated, central venous catheter-related bloodstream infections (HA-BSIs) are a major cause of morbidity and mortality. Needleless connectors (NCs) are an important component of the intravenous system. NCs initially were introduced to reduce health care worker needlestick injuries, yet some of these NCs may increase HA-BSI risk. Methods. We compared HA-BSI rates on wards or intensive care units (ICUs) at 5 hospitals that had converted from split septum (SS) connectors or needles to mechanical valve needleless connectors (MV-NCs). The hospitals (16 ICUs, 1 entire hospital, and 1 oncology unit; 3 hospitals were located in the United States, and 2 were located in Australia) had conducted HA-BSI surveillance using Centers for Disease Control and Prevention definitions during use of both NCs. HA-BSI rates and prevention practices were compared during the pre-MV period, MV period, and post-MV period. Results. The HA-BSI rate increased in all ICUs and wards when SS-NCs were replaced by MV-NCs. In the 16 ICUs, the HA-BSI rate increased significantly when SS-NCs or needles were replaced by MV-NCs (6.15 vs 9.49 BSIs per 1000 central venous catheter [CVC]-days; relative risk, 1.54; 95% confidence interval, 1.37-1.74; P< .001). The 14 ICUs that switched back to SS-NCs had significant reductions in their BSI rates (9.49 vs 5.77 BSIs per 1000 CVC-days; relative risk, 1.65; 95% confidence interval, 1.38-1.96; P<.001). BSI infection prevention strategies were similar in the pre-MV and MV periods. Conclusions. We found strong evidence that MV-NCs were associated with increased HA-BSI rates, despite similar BSI surveillance, definitions, and prevention strategies. Hospital personnel should monitor their HA-BSI rates and, if they are elevated, examine the role of newer technologies, such as MV-NCs.