Mycobacterium bovis is the causative agent of tuberculosis in cattle. Infection of macrophages with M. bovis leads to the activation of the "nucleotide binding and oligomerization, leucine-rich repeat and pyrin domains-containing protein 3" (NLRP3) and "absent in melanoma 2" (AIM2) inflammasomes, which in turn triggers release of the proinflammatory cytokine interleukin-1β (IL-1β) that contributes to bacterial clearance and plays a crucial role in the host defense. However, NLRP3 and AIM2 inflammasome activation is influenced by several factors and how IL-1β secretion by M. bovis-infected macrophages is regulated via the inflammasome pathway remains unclear. Here we found that IL-1β secretion and pro-IL-1β protein accumulation were inhibited in THP-1 macrophages upon exposure to the virulent M. bovis Beijing strain in the presence of high K(+) concentrations, cycloheximide (a protein synthesis inhibitor) and PR-619 (a deubiquitinating enzyme inhibitor). Scavenging reactive oxygen species (ROS) induced by N-acetylcysteine reduced IL-1β release independent of the mitochondrial permeability transition. Collectively, our results suggest that IL-1β secretion by M. bovis-infected THP-1 macrophages is reduced by high extracellular K(+) concentration, inhibition of new protein synthesis, deubiquitination, and ROS generation.