BackgroundThe nose and the throat are the most predominant colonizing sites ofStaphylococcus aureus, and colonization is a risk factor for infection. Nasal colonization is well described; however, we have limited knowledge aboutS. aureusthroat colonization. The main objective of this study was to explore differentially expressed genes (DEGs) inS. aureusthroat isolate TR145 exposed to human tonsil epithelial cells (HTEpiC) by using RNA sequencing (RNA-seq) and pathway analysis. DEGs inS. aureusat 1 or 3 hours (h) interaction with its host were explored.ResultsS. aureuswas co-cultured in absence and presence of tonsillar cells at 1 or 3 h. Over the 3 h time frame, the bacteria multiplied, but still caused only minor cytotoxicity. Upon exposure to tonsillar cell line,S. aureuschanged its transcriptomic profile. A total of 508 DEGs were identified including unique (1 h, 160 DEGs and 3 h, 78 DEGs) and commonly shared genes (1 and 3 h, 270 DEGs). Among the DEGs, were genes encoding proteins involved in adhesion and immune evasion, as well as iron acquisition and transport. Reverse transcription qPCR was done on selected genes, and the results correlated with the RNA-seq data.ConclusionWe have shown the suitability of using HTEpiC as an in vitro model for investigating key determinants inS. aureusduring co-incubation with host cells. Several DEGs were unique after 1 or 3 h exposure to host cells, while others were commonly expressed at both time points. As their expression is induced upon meeting with the host, they might be explored further for future targets for intervention to prevent either colonization or infection in the throat.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02919-5.