Increased physical activity (PA) is associated with improvement of cardiac risk factors and prevention of cardiovascular disease, yet many women remain sedentary. With rising Internet use, Web-based interventions provide an alternative to improve PA, but their effectiveness for change in PA and quality of life (QOL) in a real-world setting is unknown. Participants were United States women ≥18 years old who received 12 weekly PA modules and completed surveys on PA, QOL, and readiness for PA at registration (registration cohort, n = 3,796) or registration and 12 weeks (evaluation cohort, n = 892). QOL was assessed with a modified Short Form-36 with subscores for energy and well-being. Participants showed significant (p <0.001) favorable changes in PA (baseline, median 240 kcal/week, interquartile range 62 to 667; 12 weeks, 343 kcal/week, 131 to 828), stage of readiness for PA, and body mass index (baseline, 29.3 kg/m², 24.9 to 34.7; 12 weeks, 28.9 kg/m², 24.6 to 34.2). Significant improvements (p <0.0001) were also found in composite scores for energy and well-being. Compliance with PA guideline recommendations increased from 15.8% to 21.4%. Program weeks completed (p = 0.03), energy (p = 0.04), and well-being (p = 0.002) were significantly associated with achieving guideline compliance. In women reporting no PA at baseline (n = 88), program participation resulted in 54.6% achieving some PA and another 9.1% achieving total compliance with recommendations. In conclusion, in this national cohort of women, a 12-week Web-based intervention improved PA and QOL measurements, resulting in higher short-term PA guideline compliance and better QOL. Increasing use of this simple Web-based tool could improve PA and promote disease prevention.