Abnormal blood-brain barrier (BBB) is a common pathological feature in brain damage. In the present study, a brain microvascular endothelial cell (BMEC) model was established to determine the role of the toll‑like receptor 4 (TLR4)/protein kinase Cα (PKCα)/occludin signaling pathway in BBB dysfunction. Three small interfering (si)RNAs directed against PKCα were designed to investigate the molecular mechanisms of PKCα underlying BBB damage. BMECs were divided into 4 groups: Control group, TAK‑242 (a TLR4 inhibitor) group, PKCα‑siRNA group and TAK‑242+PKCα‑siRNA group. The results indicated that siRNA‑3 was the most effective at silencing PKCα gene expression. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis indicated no significant difference of TLR4 mRNA expression levels between three different treated groups and the Control group. However, PKCα mRNA expression in the PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups were significantly decreased compared with that in Control and TAK‑242 groups. In addition, occludin mRNA expression in PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups were significantly higher compared with the Control group. Meanwhile, occluding expressions in three treated groups were also significantly higher compared with the Control group. Furthermore, TAK‑242 treatment, PKCα‑siRNA treatment, and TAK‑242+PKCα‑siRNA treatment could promote occludin junctional labeling compared with the Control group. The permeability of PKCα‑siRNA and TAK‑242+PKCα‑siRNA groups was significantly promoted compared with the control group. The TLR4/PKCα/occludin signaling pathway was closely related to BBB damage. The present study will lead to an improved molecular understanding of BBB damage in the future.