Activating transcription factor 3 (ATF3) is a key transcription factor involved in regulating cellular stress responses, with different expression levels and functions in different tissues. ATF3 has also been shown to play crucial roles in regulating tumor development and progression, however its potential role in oral squamous cell carcinomas has not been fully explored. In this study, we examined biopsies of tongue squamous cell carcinomas (TSCCs) and found that the nuclear expression level of ATF3 correlated negatively with the differentiation status of TSCCs, which was validated by analysis of the ATGC database. By using gain- or loss- of function analyses of ATF3 in four different TSCC cell lines, we demonstrated that ATF3 negatively regulates the growth and migration of human TSCC cells in vitro. RNA-seq analysis identified two new downstream targets of ATF3, interferon alpha inducible proteins 6 (IFI6) and 27 (IFI27), which were upregulated in ATF3-deleted cells and were downregulated in ATF3-overexpressing cells. Chromatin immunoprecipitation assays showed that ATF3 binds the promoter regions of the IFI6 and IFI27 genes. Both IFI6 and IFI27 were highly expressed in TSCC biopsies and knockdown of either IFI6 or IFI27 in TSCC cells blocked the cell growth and migration induced by the deletion of ATF3. Conversely, overexpression of either IFI6 or IFI27 counteracted the inhibition of TSCC cell growth and migration induced by the overexpression of ATF3. Finally, an in vivo study in mice confirmed those in vitro findings. Our study suggests that ATF3 plays an anti-tumor function in TSCCs through the negative regulation of its downstream targets, IFI6 and IFI27.