Established reference standards for the ventilatory threshold (Vt) are lacking. The aim of this study was to develop reference standards for the Vt derived from cardiopulmonary exercise testing (CPX) using treadmill and cycle ergometry. Seven laboratories experienced in CPX administration with established quality control procedures contributed to the Fitness Registry and the Importance of Exercise: A National Database (FRIEND) from April 2014 through February 2019. Vt data from 27 states in the United States and the Ontario Province in Canada, comprising 9,350 tests (treadmill: n = 1,195; cycle ergometer: n = 8,155) in men (n = 7,540) and women (n = 1,810) 20 to 79 years of age who were free from smoking and known cardiovascular, pulmonary, metabolic, and/or neoplastic disease were used to develop the reference standards. Comparisons of Vt values were made between exercise testing modes, sex, and age groups. Vt values on the treadmill were higher than cycle ergometry; men had higher Vt values than women on both test modalities, and the highest Vt values achieved were in the 20- to 29-year-old age group compared with all other age groups (all P < .001). The rates of decline in Vt values from age groups 20 to 29 through 70 to 79 years of age were 23% and 35% in men and 47% and 30% in women for treadmill and cycle ergometry tests, respectively. In addition to previous reference standards from FRIEND for cardiorespiratory fitness, the Vt reference standards reported herein provide valuable information on functional metric. These data have important implications for CPX interpretation and aerobic exercise prescription in the clinical and fitness settings.