INTRODUCTION: Physical inactivity, excessive total time spent in sedentary behavior (SB) and prolonged sedentary bouts have been proposed to be risk factors for chronic disease morbidity and mortality worldwide. However, which patterns and postures of SB have the most negative impacts on health outcomes is still unclear. This population-based study aimed to investigate the independent associations of the patterns of accelerometer-based overall SB and sitting with serum lipid biomarkers at different moderate- to vigorous-intensity physical activity (MVPA) levels. METHODS: Physical activity and SB were measured in a birth cohort sample ( N = 3272) at 46 yr using a triaxial hip-worn accelerometer in free-living conditions for 14 d. Raw acceleration data were classified into SB and PA using a machine learning-based model, and the bouts of overall SB and sitting were identified from the classified data. The participants also answered health-related questionnaires and participated in clinical examinations. Associations of overall SB (lying and sitting) and sitting patterns with serum lipid biomarkers were investigated using linear regression. RESULTS: The overall SB patterns were more consistently associated with serum lipid biomarkers than the sitting patterns after adjustments. Among the participants with the least and the most MVPA, high total time spent in SB and SB bouts of 15-29.99 and ≥30 min were associated with impaired lipid metabolism. Among those with moderate amount of MVPA, higher time spent in SB and SB bouts of 15-29.99 min was unfavorably associated with serum lipid biomarkers. CONCLUSIONS: The associations between SB patterns and serum lipid biomarkers were dependent on MVPA level, which should be considered when planning evidence-based interventions to decrease SB in midlife.