Mycoplasmas bovis (M. bovis) is an important pathogen that causes a variety of diseases, such as bovine respiratory diseases and causes significant losses to the national cattle industry every year, seriously affecting the development of the cattle industry worldwide. The pathogenic mechanism of M. bovis infection is still unknown, which leads to the lack of timely diagnosis and treatment. In this study, embryonic bovine lung (EBL) cells, infected with M. bovis were collected for gene profiling and detection of marker genes in the mTOR signaling pathway. The result showed that M. bovis infection significantly inhibits EBL growth in a dose-dependent manner. The transcription profiling data uncovered that M. bovis infection repressed a series of gene expressions in EBL cells, which are mainly related to metabolic process and immune response. Notably, many marker genes in the PI3K-Akt-mTOR pathway showed down-regulation after M. bovis infection. Further evidence showed that M. bovis infection inhibits expression of mTOR signaling pathway marker genes in EBL cells, which are time dependent. To further understand the M. bovis-induced inhibitory effect of mTOR signaling pathway, this study employed FBS as a supplement for exogenous nutrients and found that addition of a high concentration of FBS can rescue M. bovis-induced cell damage. In addition, a high concentration of FBS can rescue down-regulated mTOR signaling, including increasing transcriptional expression and protein phosphorylation level of mTOR pathway marker genes. This study demonstrated that M. bovis infection leads to inhibition of the nutrient metabolic pathway mTOR in a time-dependent manner, which would be helpful to further understand M. bovis infection mechanism and develop a new efficient anti-mycoplasma strategy targeting mTOR signaling.