Purpose <p>The aim was to investigate physical activity levels and associations between physical activity levels, sedentary time, fatigue, and health-related quality of life (HRQoL) in adult survivors of childhood acute lymphoblastic leukemia (ALL).</p> Method <p>This national cross-sectional study included 426 adult survivors of childhood ALL (diagnosed 1985–2007, 49% women, median age 32 [min–max 19–49]), who completed a questionnaire on physical activity (BHW-Q), sedentary time (SED-GIH-Q), fatigue (MFI-20), and HRQoL (SF-36). Associations were analyzed using linear regression.</p> Results <p>Overall, 44% of participants did not meet physical activity recommendations. Median weekly duration of vigorous-intensity and moderate-intensity activity was 45&#xa0;min (Q1–Q3 15–105) and 120&#xa0;min (Q1–Q3 45–175), respectively. Median sedentary time was 8&#xa0;h per day (Q1–Q3 5–11). Not meeting recommendations was significantly associated with greater fatigue (total MFI-20 score β = 1.7, 95% CI 0.1 to 3.3, <i>p</i> = 0.033) and lower physical and mental health components of HRQoL (SF-36 PCS β = -3.3, 95% CI -5 to -1.5, <i>p</i> &lt; 0.001; SF-36 MCS β = -4.2, 95% CI -6 to -1.8, <i>p</i> &lt; 0.001). Sedentary time ≥ 7&#xa0;h per day was significantly associated with lower mental HRQoL (SF-36 MCS β = -3.6, 95% CI -5.9 to -1.2, <i>p</i> = 0.003).</p> Conclusion <p>Many adult survivors of childhood ALL had insufficient levels of physical activity and accumulated considerable sedentary time. Insufficient physical activity was associated with greater fatigue and lower HRQoL, while greater sedentary time was associated with lower mental HRQoL. However, given the cross-sectional design, causality cannot be inferred.</p> Implications for Cancer Survivors <p>Physical activity promotion is a potential strategy to reduce fatigue and enhance HRQoL among adult survivors of childhood ALL.</p>