[Objective] Effect of mycoplasma on gene function was studied in vitro. [Methods] FIGNL1, a critical gene to repair DNA double-strand breaks, was suppressed by siRNA in H446 and H1688 cells infected with or without Mycoplasma hyorhinis or after mycoplasma removal. Expression of targeted gene and cell cycle analysis was measured using real-time PCR, flow cytometry and other methods. [Results] No significant effect of M. hyorhinis was observed on the suppression of FIGNL1 expression by siRNA. After expression of FIGNL1 was suppressed in H1688 and H446 cells without Mycoplasma infection or after Mycoplasma removal, no significant change of cells proportion in S phase was observed between experimental group targeting FIGNL1 (T1) and negative control group compared with the blank group containing transfection reagent (mock). However, cell proportion in S phase of experimental and negative control group was increased approximately 1.38 and 0.51 folds, respectively comparing with blank group in H1688 cells infected with M. hyorhinis, 1.27 and 0.55 folds respectively in H446 cells infected with M. hyorhinis. [Conclusion] When the expression of FIGNL1 was suppressed in H1688 and H446 cells, M. hyorhinis can significantly induce S phase arrest because Mycoplasma can cause host cells DNA damage and FIGNL1 is a critical gene to repair DNA double-strand breaks. Mycoplasma is widespread and significantly influential on cells, we should be highly alert to it on function study of gene and tumor research.