Background: Recent updates to physical activity guidelines highlight the importance of reducing sedentary time. However, at present, only general recommendations are possible (ie, “Sit less, move more”). There remains a need to investigate the strength, temporality, specificity, and dose–response nature of sedentary behavior associations with chronic disease, along with potential underlying mechanisms. Methods: Stemming from a recent research workshop organized by the Sedentary Behavior Council themed “Sedentary behaviour mechanisms—biological and behavioural pathways linking sitting to adverse health outcomes,” this paper (1) discusses existing challenges and scientific discussions within this advancing area of science, (2) highlights and discusses emerging areas of interest, and (3) points to potential future directions. Results: A brief knowledge update is provided, reflecting upon current and evolving thinking/discussions, and the rapid accumulation of new evidence linking sedentary behavior to chronic disease. Research “action points” are made at the end of each section—spanning from measurement systems and analytic methods, genetic epidemiology, causal mediation, and experimental studies to biological and behavioral determinants and mechanisms. Conclusion: A better understanding of whether and how sedentary behavior is causally related to chronic disease will allow for more meaningful conclusions in the future and assist in refining clinical and public health policies/recommendations.