Tembusu virus (TMUV) causes disease in poultry, especially in ducks, resulting in abnormality in egg production and with high morbidity and mortality, resulting in great loss in duck farming industry in China and Southeast Asia. Previous studies on the pathogenesis of TMUV infection have been mostly conducted in poultry, with a few studies being undertaken in mice. While TMUV does not cause disease in humans, it has been reported that antibodies against TMUV have been found in serum samples from duck farmers, and thus data on TMUV infection in humans is limited, and the pathogenesis is unclear. In this study we investigated the cell tropism and potential susceptibility of humans to TMUV using several human cell lines. The results showed that human nerve and liver cell lines were both highly susceptible and permissive, while human kidney cells were susceptible and permissive, albeit to a lower degree. In addition, human muscle cells, lung epithelial cells, B-cells, T-cells and monocytic cells were largely refractory to TMUV infection. This data suggests that liver, neuron and kidney are potential target organs during TMUV infection in humans, consistent with what has been found in animal studies.