Japanese encephalitis virus (JEV) is known as an important mosquito-borne human pathogen that causes Japanese encephalitis and may lead to lethal effect. Since monocyte has been demonstrated to play transmissible role for JEV, rare study is reported to clarify the effect of JEV envelope (JEVE) protein on monocyte. This study intends to investigate the effects of JEVE protein inside monocyte. Notably, significant decreased expression of tumour necrosis factor (TNF)-α mRNA in RAW264.7 cells transfected with pEGFP-JEVE was observed as compared to those cells transfected with pEGFP. Increased p21Waf1/Cip1 protein was observed in both pEGFP and pEGFP-JEVE transfected RAW264.7 cells. However, increased p53 protein was only detected in pEGFP-transfected cells but not pEGFP-JEVE transfected cells as well as the result that no increased expression of nuclear factor-kB was observed in pEGFP-JEVE transfected cells. These experimental results indicate the effects of JEVE protein in alleviating TNF-α mRNA expression that is associated with the increased p53-independent p21Waf1/Cip1 expression and provide an explanation in the role of JEV transmission through monocyte.