Doxorubicin remains an important part of chemotherapy regimens in the clinic and is considered an effective agent in the treatment of acute lymphoblastic leukemia (ALL). Although the cellular responses induced by doxorubicin treatment have been investigated for years, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we utilized mass spectrometry, together with stable isotope labeling by amino acids in cell culture (SILAC), to analyze comparatively the protein expression in Jurkat-T cells before and after treatment with a clinically relevant concentration of doxorubicin. We were able to quantify 1066 proteins in Jurkat-T cells with both forward and reverse SILAC measurements, among which 62 were with significantly altered levels of expression induced by doxorubicin treatment. These included the up-regulation of core histones, heterogeneous nuclear ribonucleoproteins, and superoxide dismutase 2 as well as the down-regulation of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase. The latter two are essential enzymes for cholesterol biosynthesis. We further demonstrated that the doxorubicin-induced growth inhibition of Jurkat-T cells could be rescued by treatment with cholesterol, supporting that doxorubicin exerts its cytotoxic effect, in part, by suppressing the expression of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase, thereby inhibiting the endogenous production of cholesterol. The results from the present study provide important new knowledge for gaining insights into the molecular mechanisms of action of doxorubicin.