This study aimed to provide a quantitative synthesis of the effect of Self-determination theory (SDT) based instructional interventions on the motivational regulations of participants in organized physical activity. We conducted a systematic review and meta-analysis on experimental studies conducted before December 2021. The search using the online databases PsychINFO, PsychARTICLES, ERIC, SportDISCUS, and ProQuest Dissertations and Theses, and Google Scholar and other supplementary search strategies yielded 7774 articles, with 38 articles (142 effects and 12,457 participants) meeting the inclusion criteria. The articles were analyzed using a metaanalytic multivariate model. The study showed that SDT-based instruction had a positive heterogeneous small effect on intrinsic motivation (g = 0.29; CI 95% [0.17, 0.41]) and identified regulation (g = 0.23; CI 95% [0.10, 0.35]) and a negative heterogeneous, small effect on external regulation (g = -0.16; CI 95% [-0.31, -0.00]) and amotivation (g = -0.14; CI 95% [-0.28, -0.01]). SDT-based instruction did not have an effect on integrated regulation (g = 0.08; CI 95% [-0.11, 0.28]) nor introjected regulation (g = 0.03; CI 95% [-0.7, 0.13]). Univariate categorical moderator analyses highlighted multiple variables that impacted the size of the effects on the outcomes, including type of intervention and control group, length of study, age of participants, and study quality. Findings from the moderator analyses challenge the practical implications of SDT-based instructional interventions in improving motivation in organized physical activity. High-quality experimental trials using careful and precise conceptualizations of need-supportive behaviors and strategies would benefit the discipline.