Porcine reproductive and respiratory syndrome virus (PRRSV) is the leading pathogen in swine industry. Our laboratory recently demonstrated that unidentified low molecular weight molecules in the culture supernatant of Actinobacillus pleuropneumoniae can block in vitro replication of PRRSV in SJPL infected cells. The characterization of this inhibitory activity can provide clues for the development of new prophylactic and therapeutic treatments against PRRSV. Therefore, the aim of this investigation was to study the antiviral activity of A. pleuropneumoniae. We hypothesised that A. pleuropneumoniae can induce cell cycle disruption of SJPL cells. To test our hypothesis, SJPL cells were treated with A. pleuropneumoniae culture supernatant. The cell cycle and cell proliferation were then analyzed. First, cell cycle analysis using flow cytometry indicated that the A. pleuropneumoniae supernatant significantly shifted the cell population towards an arrest in the G2/M phase (n=15; p≤0.0001). Second, we observed that the A. pleuropneumoniae supernatant significantly reduced cell proliferation (n=8; p≤0.05). To further test our hypothesis, SJPL cells were infected with PRRSV and treated with 3,3'-methylenediindole (DIM), a molecule known to cause an arrest in the G2/M phase and a reduction in cell proliferation. Cells treated with this molecule did not support PRRSV replication, suggesting that disruption of the cell cycle and cell proliferation can inhibit the replication of PRRSV. The proteomes of SJPL cells that were either treated or not with A. pleuropneumoniae culture supernatant were then compared using Westernblot analysis. A modulation of proteins implicated in the regulation of the cell cycle was indeed observed. In conclusion, the modulation of proteins associated with the regulation of the cell cycle of SJPL cells causes an inhibition of PRRSV replication.