Pancreatitis-associated protein 2 (PAP2) is a member of the Reg3 gene family and is classified as a group 7 C-type lectin-like protein. In rats, each of the three PAP isoforms has independent immunologic functional effects on macrophages. We have previously shown that PAP2 up-regulates inflammatory cytokines in macrophages in a dose-dependent manner and acts through NF-kappaB mechanisms. In this study, we aimed to determine protein domains that are essential for the immunologic function of PAP2 by mutational or chemical analysis. The protein activity for each mutant was determined by measuring TNF-alpha, IL-6, or IL-1 production in macrophages. Truncation of the first 25 residues on the N terminus of PAP2 did not affect protein activity whereas truncation of the last 30 residues of the C terminus of PAP2 completely inactivated the function of PAP2. Additionally, reduction of three disulfide bonds proved to be important for the activity of this protein. Further investigation revealed two invariant disulfide bonds were important for activity of PAP2 while the disulfide bond that is observed in long-form C-type lectin proteins was not essential for activity. Coupling the ability of PAP2 to up-regulate inflammatory cytokines via NF-kappaB with its associated expression in acute pancreatitis, a condition with aberrant concentrations of inflammatory cytokines, we investigated whether PAP2 mutants mechanistically activate the NF-kappaB-signaling pathway and demonstrate that preincubation with select rPAP2 mutant proteins affect translocation of this transcription factor into the nucleus.