Highlights► HIV-1 p6 exhibits N- and C-terminal helical domains in micelle solution at pH 7. ► p6 interacts with a cytoplasmic model membrane through N- and C-terminal regions. ► Phosphorylation of Ser-40 does not alter the secondary structure of the protein. ► Phosphorylation of Ser-40 amplifies the interaction of p6 with membranes. ► Effects of p6 S40F mutation indicate p6 modulates membrane interactions of HIV Gag.