HIV-1 Vpu antagonizes the block to particle release mediated by CD317 (BST-2/HM1.24/Tetherin) via incompletely understood mechanisms. Vpu and CD317 partially reside in cholesterol-rich lipid rafts where HIV-1 budding preferentially occurs. Here we find that lipid raft association of ectopically expressed or endogenous CD317 was unaltered upon co-expression with Vpu or following HIV-1 infection. Similarly, Vpu's lipid raft association remained unchanged upon expression of CD317. We identify amino acids V25 and Y29 of Vpu as crucial for microdomain partitioning and single substitution of these amino acids resulted in Vpu variants with markedly reduced or undetectable lipid raft association. These mutations did not affect Vpu's subcellular distribution and binding capacity to CD317, nor its ability to downmodulate cell surface CD317 and promote HIV-1 release from CD317-positive cells. We conclude that (i) lipid raft incorporation is dispensable for Vpu-mediated CD317 antagonism and (ii) Vpu does not antagonize CD317 by extraction from lipid rafts.