To examine the role of caspase-1 and the NLRC4 inflammasome during bacterial infection, C57BL/6, IL-1β(-/-), caspase-1(-/-), and NLRC4(-/-) mouse corneas were infected with ExoS/T- or ExoU-expressing Pseudomonas aeruginosa. We found that IL-1β was essential for neutrophil recruitment and bacterial clearance and was produced by myeloid cells rather than resident cells. In addition, neutrophils were found to be the primary source of mature IL-1β during infection, and there was no significant difference in IL-1β processing between C57BL/6 and caspase-1(-/-) or NLRC4(-/-) infected corneas. IL-1β cleavage by human and mouse neutrophils was blocked by serine protease inhibitors and was impaired in infected neutrophil elastase (NE)(-/-) corneas. NE(-/-) mice also had an impaired ability to clear the infection. Together, these results demonstrate that during P. aeruginosa infection, neutrophils are the primary source of mature IL-1β and that IL-1β processing is dependent on serine proteases and not NLRC4 or caspase-1.