Dehydroepiandrosterone (DHEA) is a natural hormone with many beneficial properties including an anticancer activity. Unfortunately, DHEA is unstable in the body and exhibits cytotoxicity against healthy cells. In this study, a series of new phosphocholines containing DHEA at sn-1 and/or sn-2 positions were prepared. Succinic acid was used as a linker between the active drug and sn-glycero-3-phosphocholine. All the compounds were evaluated in vitro for their antiproliferative activities against four cell lines: Balb/3T3, HL-60, B16, and LNCaP. The results showed that phosphocholines with DHEA at sn-1 and/or sn-2 positions did not have cytotoxic effects on the normal cell line (Balb/3T3). Mixed-chain phospholipids with DHEA and fatty acid residues showed the highest activity against tumor cell lines. The most active compound, 11c, showed a moderate cytotoxic effect against the HL-60 and B16 cell lines.