P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.