Phosphatidylserine (PS) is a negatively-charged glycerophospholipid found mainly in the plasma membrane (PM) and in the late secretory/endocytic compartments, where it regulates cellular activity and can mediate apoptosis. Export of PS from the endoplasmic reticulum, its site of synthesis, to other compartments, and its transbilayer asymmetry must therefore be precisely regulated. We review recent findings on non-vesicular transport of PS by lipid transfer proteins (LTPs) at membrane contact sites, on PS flip-flop between membrane leaflets by flippases and scramblases, and on PS nano-clustering at the PM. We also discuss emerging data on cooperation between scramblases and LTPs, how perturbation of PS distribution can lead to disease, and the specific role of PS in viral infection.