The antimicrobial activity of exocrine pancreatic juice is an important component of gastrointestinal tract innate defenses, yet little is known about whether secretion is regulated in parallel with digestive enzymes. In this study, we used 8 pigs with pancreatic catheters to quantify antibacterial activity and measure protein content (indicator of enzyme secretion) of pancreatic juice collected hourly from 0700 to 1900, with the animals adapted to being fed at 0800 and 1600. Antibacterial activity in the samples of pancreatic juice was quantified by comparing the growth inhibition of Staphylococcus aureus subsp. aureus strain ATCC 6538P relative to a known concentration of gentamicin. Antibacterial activity (U/mL and /min) was highest in samples collected 1 hour prior to feeding (equivalent to 0.6 microgram gentamicin/mL), declined as the meal was consumed, and was lower (P < 0.05) in samples collected while the meals were being digested (0.41 microgram gentamicin/mL). Protein content was negatively correlated with antibacterial activity, with protein secretion lower (mg/mL and /min) before feeding, with an increase as the pigs ate and digested the meals (P < 0.05). The results indicate that the antibacterial activity in pancreatic juice is not secreted in parallel with protein secretion, suggesting that regulation involves alternative signaling pathways or contrasting responses to shared signals.