Background <p><i>Neospora caninum</i> is an apicomplexan parasite responsible for bovine neosporosis, a disease that leads to substantial economic losses in cattle due to abortion and reduced productivity. The pathogenesis of <i>N</i>. <i>caninum</i> is shaped by complex host–parasite interactions, and virulence is known to vary between strains. BPK1 (Bradyzoite pseudokinase 1), a pseudokinase previously identified as a potential virulence factor in <i>Toxoplasma gondii</i>, has not yet been functionally characterized in <i>N</i>. <i>caninum</i>.</p> Methods <p>To investigate the role of NcBPK1 in parasite virulence, a knockout strain (<i>Nc</i>Δ<i>BPK1</i>) was generated using CRISPR/Cas9 genome editing. The virulence of the mutant was evaluated in a pregnant mouse model by assessing neonatal survival and parasite burden in dam tissues. In vitro assays were conducted to examine parasite replication in bovine macrophages and to analyze the expression of stage-specific genes.</p> Results <p>Deletion of <i>NcBpk1</i> resulted in enhanced parasite virulence in vivo, as shown by a decrease in neonatal survival and higher parasite loads in maternal brain tissue. The <i>Nc</i>Δ<i>BPK1</i> mutant also displayed enhanced replication in bovine macrophages and reduced expression of bradyzoite-specific genes, suggesting a defect in stage conversion.</p> Conclusions <p>These findings indicate that NcBPK1 is crucial for regulating the balance between acute replication and chronic persistence. Its absence promotes rapid tachyzoite proliferation and worsens disease outcomes. This study sheds light on the molecular mechanisms underlying <i>N</i>. <i>caninum</i> virulence. Further research is needed to elucidate the signaling pathways and protein interactions involving NcBPK1.</p> Graphical Abstract <p></p>