<p class="MsoNormal" style="line-height: 150%;"><strong>Background</strong> Sedentary behavior is recognized as a distinct construct from lack of moderate-vigorous physical activity and is associated with deleterious health outcomes. Previous studies have primarily relied on self-reported data, while data on the relationship between objectively-measured sedentary time and cardiometabolic biomarkers are sparse, especially among U.S. Hispanics/Latinos.</p><p class="MsoNormal" style="line-height: 150%;"><strong>Methods and Results </strong>We examined associations of objectively-measured sedentary time (via Actical accelerometers for 3-7 days) and multiple cardiometabolic biomarkers among 12,083 participants, aged 18-74 years, from the Hispanic Community Health Study/Study of Latinos. Hispanics/Latinos of diverse backgrounds (Central American, Cuban, Dominican, Mexican, Puerto Rican, and South American) were recruited from 4 U.S. cities between 2008 and 2011.&nbsp; Sedentary time (&lt;100 counts/minute) was standardized to 16-hour/day of wear time. The mean sedentary time was 11.9 hours/day (74% of accelerometer wear time). After adjustment for moderate-vigorous physical activity and confounding variables, prolonged sedentary time was associated with decreased high-density lipoprotein (HDL)-cholesterol (<em>P</em>=0.04), and increased triglycerides, 2-hour glucose, fasting insulin and HOMA-IR (all <em>P</em>&lt;0.0001). These associations were generally consistent across age, sex, Hispanic/Latino backgrounds, and physical activity levels. Notably, even among individuals meeting physical activity recommendations, sedentary time was detrimentally associated with several cardiometabolic biomarkers (diastolic blood pressure, HDL-cholesterol, fasting and 2-hour glucose, fasting insulin and HOMA-IR; all <em>P</em>&lt;0.05).</p><p>&nbsp;</p><p class="MsoNormal" style="line-height: 150%; mso-layout-grid-align: none; text-autospace: none;"><strong>Conclusions </strong>Our large population-based, objectively-derived data showed deleterious associations between sedentary time and cardiometabolic biomarkers, independent of physical activity, in U.S. Hispanics/Latinos. Our findings emphasize the importance of reducing sedentary behavior for the prevention of cardiometabolic diseases, even in those who meet physical activity recommendations.</p>