Mycoplasma hyopneumoniae (M. hyopneumoniae) infection affects the swine industry. Lithium chloride (LiCl), is a drug used to treat bipolar disorder and has also shown activity against bacterial and viral infections. Herein, we evaluated the antibacterial activity of LiCl on PK-15 cells infected with M. hyopneumoniae. Incubation of LiCl (40mM) with cells for 24h, did not significantly affect the cell viability. The qRT–PCR showed ~80% reduction in M. hyopneumoniae genome when LiCl added post-infection. A direct effect of LiCl on bacteria was also observed. However, treatment of cells with LiCl prior infection, does not protect against the infection. Anti-bacterial activity of LiCl was further confirmed by IFA, which demonstrated a reduction in the bacterial protein. With 40mM LiCI, the apoptotic cell death, production of nitric oxide and superoxide anion induced by M. hyopneumoniae, were prevented by ~80%, 60% and 58% respectively. Moreover, caspase-3 activity was also reduced (82%) in cells treated with 40mM LiCl. LiCl showed activity against various strains of M. hyopneumoniae examined in our study. Collectively, our data showed that LiCl inhibited the infection of M. hyopneumoniae through anti-apoptotic mechanism. •LiCl inhibits Mycoplasma hyopneumoniae infection in PK-15 cells dose-dependent manner.•LiCl inhibits 80% of apoptosis induced M. hyopneumoniae infection in PK-15 cells.•LiCl protects against the infection of various strains of M. hyopneumoniae infected PK-15 cells.