This study examines how different concentrations of arginine vasopressin (AVP) and its preservative chlorobutanol (ClB) impact the immune functions of human polymorphonuclear neutrophils (PMNs), which are crucial in the immune response, particularly in sepsis. Using a model to simulate the physiological, sepsis-related, and therapeutic AVP levels in plasma, we analysed how AVP and ClB affect PMN activities, including reactive oxygen species (ROS) production, NETosis, antigen expression, and migration. PMNs were isolated from whole human blood and assessed using flow cytometry and live cell imaging. The results indicated that neither AVP nor ClB significantly affected PMN viability, antigen expression, NETosis, or ROS production in response to N-Formylmethionine-leucyl-phenylalanine, or fMLP, and tumour necrosis factor alpha. In the migration assays, concentration-dependent effects were observed. At physiological AVP levels, PMN migration showed no reduction, while the sepsis-associated AVP levels initially reduced migration before returning to the baseline or even increasing. The therapeutic AVP concentrations showed similar migration to that in the controls, while high concentrations progressively inhibited migration. ClB, regardless of its concentration, enhanced PMN migration. These findings suggest that AVP during sepsis may impair PMN migration, potentially contributing to tissue damage and systemic complications. This highlights AVP’s role as a possible immune modulator in complex immune responses.