Abstract Context Vitamin D deficiency is considered a global public health issue, even in low-latitude countries where there is abundant sunlight radiation. Nevertheless, the prevalence of vitamin D insufficiency and deficiency in the South American continent has not been well characterized. Objective The aim of this review was to estimate the prevalence of vitamin D deficiency (25-hydroxy-calciferol < 20 ng/mL) in South American populations. Data Sources Seven electronic databases (MEDLINE, Web of Science, Embase, Biblioteca Virtual de Saúde, SciELO, Scopus, and Google Scholar) were searched systematically for observational studies published before July 1, 2021, and reporting vitamin D status of healthy adults in South America. Data Extraction Data were extracted using a standardized form. Risk of bias was assessed by the Joanna Briggs Institute Critical Appraisal Instrument for Studies Reporting Prevalence. All steps were conducted independently by two authors. Data were pooled using a random-effects model. Stratified meta-analysis and meta-regression were conducted (R software). Data Analysis Of 9460 articles identified, 96 studies with a total of 227 758 participants were included. The overall prevalence of vitamin D deficiency was 34.76% (79 studies; 95%CI, 29.68–40.21; I2 = 99%). There were significant differences in prevalence rates related to age, sex, country, latitude, season, and year of publication. Conclusion The prevalence of vitamin D deficiency is unexpectedly elevated in South American populations. Public health strategies should include efforts to prevent, detect, and treat vitamin D deficiency. Systematic Review Registration PROSPERO registration number CRD42020169439.