The effects of the human immunodeficiency virus type 1 (HIV‐1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline‐repressible expression system. Expressed Tat protein (aa 1–101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin‐induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat‐Tat cells. Protein identification was performed using 2‐D DIGE followed by MS analysis. We identified the down‐regulation of several cytoskeletal proteins such as actin, β‐tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho‐GDI complex. Down‐expression of these proteins could be involved in the survival of long‐term reservoirs of HIV‐infected CD₄⁺ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.