Blood-brain barrier (BBB) is the entry point for many neurotropic viruses and has an active role in the antiviral innate immunity of the central nervous system (CNS). We previously showed that human brain microvascular endothelial cells (HBMEC) express a variety of pattern recognition receptors (PRRs) for HIV-derived pathogen associated molecular patterns (PAMPs), including toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I). TLR3/RIG-I activation of HBMEC induced the secretion of antiviral IFNs and IFN-stimulated genes (ISGs), contributing to the suppression of HIV in macrophages. In the present study, we examined the effect of recombinant human IFN-λ (rhIFN-λ) on the viral sensing and controlling of HBMEC in the context of HIV infection. The PRRs expression of HBMEC was measured by flow cytometry and western blot. The expression of ISGs and tight junction proteins was detected by quantitative RT-PCR and western blot. We demonstrated that rhIFN-λ increased the expression of TLR3, RIG-I, and TLR7/8 in primary HBMEC. IFN-λ priming of HBMEC enhanced the induction of ISGs in response to challenge with dsRNA mimic polyinosinic:polycytidylic acid (Poly I:C) and the U-rich single-stranded RNA (ssRNA40) derived from HIV long terminal repeat (LTR). The elevated TLR3 expression was responsible for the enhanced ISG induction as TLR3 siRNA reversed the effect. More importantly, the exosomes released from rhIFN-λ primed and Poly I:C-stimulated HBMEC culture contained increased levels of ISGs, which contribute to anti-HIV activity transmission to macrophages. Consistent with the tightening of BBB in mice model, rhIFN-λ treatment also increased the expression of tight junction proteins ZO-1 and occludin of primary HBMEC. These findings provide the experimental evidence that IFN-λ participates in the BBB innate immunity by evoking the antiviral response of bystander HBMEC against HIV neuroinvasion.