Toxoplasma gondii, an opportunistic pathogenic protozoan, exhibits a strong predilection to infect the brain, causing severe neurological diseases, such as toxoplasmic encephalitis (TE), in immunocompromised patients. Microglia, the resident immune cells in the brain, is reported to play important roles in regulating the neuroinflammation mediated by T. gondii infection. Here we demonstrated that the tachyzoites of T. gondii RH strain could significantly upregulate the expression levels of microglial M1 phenotype markers including IL-1β, IL-6, TNF-α, iNOS and IL18 in activated murine BV2 microglia cells, which were regulated by T. gondii rhoptry protein 18 (TgROP18). Moreover, we found that TgROP18 could enhance the expression of M1 phenotype markers in activated murine BV2 microglia cells via activating NF-κB signal pathway. Additionally, TgROP18 was suggested to interact with the host p65 in activated murine BV2 microglia cells and induce the phosphorylation of p65 at S536. In summary, the present study demonstrated that TgROP18 could promote the activated microglia to polarize to M1 phenotype and enhanced the expression of pro-inflammatory factors via activating NF-κB signal pathway, which could contribute to elucidating the mechanism underlying the neuroinflammation mediated by activated microglia in the brain with T. gondii infection.