Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti‐HIV herbal drugs has proceeded slowly, partly because of the lack of a high‐throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti‐HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C‐X‐C receptor 4 (CXCR4) and C‐C receptor 5 (CCR5) promoter activities. A single‐round pseudotyped HIV‐luciferase reporter virus system (HIV‐Luc) was used to identify potential anti‐HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV‐1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down‐regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 µm) inhibited HIV‐1 in a dose‐dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers (p < 0.05). Spica Prunellae and Herba Andrographitis (EC50: 3.18 and 5.49 µg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti‐HIV‐1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV‐1 replication. Copyright © 2011 John Wiley & Sons, Ltd.