Updated project metadata. Despite the importance of bovine neosporosis, important knowledge gaps remain concerning pathogenic mechanisms of N. caninum. The infection of the placenta is a crucial event in the pathogenesis of the disease, however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3, involving proliferative capacity and transcriptome host cell modulation. Herein, we use the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analyzed the proteome differences between high (Nc-Spain7)- and low (Nc-Spain1H)- virulence isolates. The results showed mitochondrial processes and metabolism as the main point of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed the highest level of influence on host cell proteome in contrast to Nc-Spain7 infection. These results confirm Nc-Spain7 capacities to evade host cell response and establish infection more effectively.