Interest in manned spaceflights has increased in recent years and this brings about a surge in the need for more in-depth research into the adverse effects of spaceflights on the human body. In the absence of gravitational forces there are countless negative effects on the immune system. This dysfunction in the immune system can lead to increased susceptibility of infections by astronauts and poor wound healing. In particular, there is a lack of research on the effects of microgravity on immune cells in physiologically relevant cellular microenvironments. Amongst the various immune cell types, dendritic cells (DCs) are the primary mediators between the innate and adaptive immune systems and any dysregulation of these cells can lead to inadequate immune responses, especially in a long-term specific immune response. To gain insight into how DCs’ function is affected under microgravity conditions on Earth, we utilized loose and dense 3D fibrillar collagen matrices to explore the effects of simulated microgravity using Random Positioning Machine (RPM) on cells of the immune system. Immune potency of DCs was assessed in terms of their transcriptome profile, differentiation state, secreted cytokine profile, antigen uptake, and their ability to trigger a T-lymphocyte cell response. The transcriptome profile, using RNA-sequencing, showed that DC differentiation and maturation were altered under simulated microgravity conditions in a matrix density dependent manner. In addition, surface markers, cytokine secretion profile, and functional assays of DCs were reduced upon exposure to simulated microgravity conditions. Overall, our work pinpoints the importance of mechanotransduction in DC differentiation and function under simulated microgravity conditions, which could contribute to the design of immune modulating materials for use in spaceflight. In addition, as microgravity-associated physiological alterations closely resemble those found in the elderly, this data is not only important for space biology but could also be beneficial for a simulated model of aging on Earth.