The role of cholinesterase in inflammatory reactions has been described in several infectious diseases. However, in Brucella spp. this has not yet been studied. Therefore, the objective of this study was to evaluate whether experimental infection by Brucella ovis alters the cholinergic activity in pro- or anti-inflammatory responses to the disease. For the study 48 mice were used, 24 infected by B. ovis and 24 non-infected. We collected samples of whole blood on days 7, 15, 30 and 60 post-infection (PI) by B. ovis. Acetylcholinesterase (AChE) activity in the blood increased on days 15 and 60 PI (P < 0.05). Butyrylcholinesterase (BChE) activity in serum increased on days 7 and 60 PI (P < 0.05). An increase in serum free radical levels occurred on days 7, 15 and 60 PI (P < 0.05), and consequently superoxide dismutase activity increased on day 15 PI (P < 0.05). A reduction in catalase activity occurred when the infection became chronic (60 PI). The increase in AChE and BChE characterized a pro-inflammatory response, since these enzymes regulate levels of acetylcholine (ACh) and butyrylcholine (BuSCh), molecules with anti-inflammatory properties. Therefore, with the increase of cholinesterase activity, there was an extracellular reduction of ACh, an inhibitor of several inflammatory mediators. This proinflammatory response of B. ovis infection leads to oxidative stress, and consequently to cellular damage.