Human immunodeficiency virus (HIV) testing is the first step in the continuum of HIV prevention, care, and treatment services, without which, gaps in HIV diagnosis cannot be addressed. National HIV testing campaigns are useful for promoting HIV testing among large numbers of persons. However, the impact of such campaigns on identification of new HIV-positive diagnoses is unclear. To assess whether National HIV Testing Day (NHTD, June 27) was effective in identifying new HIV-positive diagnoses, National HIV Prevention Program Monitoring and Evaluation (NHM&E) data for CDC-funded testing events conducted during 2011-2014 were analyzed. The number of HIV testing events and new HIV-positive diagnoses during June of each year were compared with those in other months by demographics and target populations. The number of HIV testing events and new HIV-positive diagnoses were also compared for each day leading up to and after NHTD in June and July of each year. New HIV-positive diagnoses peaked in June relative to other months and specifically on NHTD. During 2011-2014, NHTD had a substantial impact on increasing the number of persons who knew their HIV status and in diagnosing new HIV infections. NHTD also proved effective in reaching persons at high risk disproportionately affected by HIV, including African American (black) men, men who have sex with men (MSM), and transgender persons. Promoting NHTD can successfully increase the number of new HIV-positive diagnoses, including HIV infections among target populations at high risk for HIV infection.