Background: It is unclear how levels of sport participation have changed among adolescents in the United States (US) in recent years, particularly considering evidence of increasing sport professionalization and attrition. Purpose: To evaluate the differences and trends in single- and multiple-team sport participation among US high school students between 1999 and 2019. Study Design: Descriptive epidemiology study. Methods: Using the Youth Risk Behavior Surveillance System of the US Centers for Disease Control and Prevention, a biannual representative survey of US high school students, data on self-reported sport participation (no participation, single team, multiple teams) within the previous year were evaluated. Differences in sport participation prevalence between 1999 and 2019 were assessed in sex strata by grade, race/ethnicity, and US region. Weighted trends using the National Center for Health Statistics Guidelines for Analysis of Trends and logistic regression models were employed for analyses. Results: The study sample over the 20-year period (N = 149,404) was 49.8% female; the majority were in ninth grade (27.7% female, 28.7% male), were non-Hispanic White (56.1% female, 56.8% male), and resided in the Southern US (57.6% female, 57.4% male). Among girls, 27.2% (95% CI, 25.8-28.6) reported multiple sports team participation in 1999, while 21.3% (95% CI, 20.1-22.6) reported the same in 2019. Among boys, 39.5% (95% CI, 38.4-40.7) reported multiple sports team participation in 1999 versus 29.1% (95% CI, 27.7-30.6) in 2019. Analyses of trends indicated that girls experienced increasing trends in single-sport team participation (β = 0.04, SE = 0.01, P < .001). Among boys, there were increasing trends in no sport participation (β = 0.06, SE = 0.01, P < .001), while multiple-sport team participation decreased over the 20-year period (β = −0.10, SE = 0.01, P < .001). Disparities in trends were observed among female and male groups by race/ethnicity, grade, and region. Conclusion: Sport participation over a 20-year period in the US decreased significantly for boys and increased for single-sports teams among girls. Sport specialization may be contributing some of the observed trends, though limitations in the data require additional research to confirm these hypotheses. Tailored efforts to increase sport participation among both sexes is needed.