Adaptive immunity is critical for distinguishing self from non-self threats. However, how our immune system is able to respond to dangers and spare self entities remains a subject of immense research. Due to its non-polymorphic nature and its capability to present diverse lipids to T cells, featuring polarizing immunomodulatory functions, the antigen-presenting molecule CD1d has been a focal point in vaccine development in the last decade. Consequently, there is a fair understanding of the relationship between the types of lipids presented by CD1d and its associated T cell-mediated biological functions. The molecular mechanisms in antigen-presenting cells (APCs) governing the presentation of these antigens to T cells however remain relatively less known, yet they are important for guiding the antigen-related effector functions of T cells. Here, using advanced live-cell optical microscopy, we studied the role of the intracellular cortical actin cytoskeleton in CD1d-mediated lipid antigen presentation. We used the drugs known to specifically interfere with actin polymerization and compared the effect they had on the lateral diffusion of plasma membrane CD1d and on their efficiency to activate T cells. These experiments uncovered a crucial role for the actin cortical meshwork in APCs in fine-tuning lipid antigen presentation. We also found a decrease in CD1d mobility upon lipid antigen loading which is potentially a result of an accompanying extensive reorganization of actin cytoskeleton.