The Trans-Activator of Transcription (Tat) of Human Immunodeficiency Virus (HIV-1) is involved in virus replication and infection and can promote oxidative stress in human astroglial cells. In response, host cells activate transcription of antioxidant genes, including a subunit of System Xc−cystine/glutamate antiporter which, in turn, can trigger glutamate-mediated excitotoxicity. Here, we present data on the efficacy of bovine Lactoferrin (bLf), both in its native (Nat-bLf) and iron-saturated (Holo-bLf) forms, in counteracting oxidative stress in U373 human astroglial cells constitutively expressing the viral protein (U373-Tat). Our results show that, dependent on iron saturation, both Nat-bLf and Holo-bLf can boost host antioxidant response by up-regulating System Xc−and the cell iron exporter Ferroportin via the Nuclear factor erythroid 2-related factor (Nrf2) pathway, thus reducing Reactive Oxygen Species (ROS)-mediated lipid peroxidation and DNA damage in astrocytes. In U373-Tat cells, both forms of bLf restore the physiological internalization of Transferrin (Tf) Receptor 1, the molecular gate for Tf-bound iron uptake. The involvement of astrocytic antioxidant response in Tat-mediated neurotoxicity was evaluated in co-cultures of U373-Tat with human neuronal SH-SY5Y cells. The results show that the Holo-bLf exacerbates Tat-induced excitotoxicity on SH-SY5Y, which is directly dependent on System-Xc−upregulation, thus highlighting the mechanistic role of iron in the biological activities of the glycoprotein.