Lactoferrin is thought to be the most polyvalent protein present in host defense against tissue injuries and infections in vertebrates. Owing to the propensity of its basic N-terminal domain to interact with various microbial and host targets, lactoferrin not only has antimicrobial properties, but also modulates the innate and adaptive immune responses. Lactoferrin may indeed up- and downregulate immune cell activation, migration, and growth. Whereas the immunomodulatory properties of lactoferrin are evidenced from in vivo studies using either lactoferrin-knockout, lactoferrin-overexpressing transgenic models, and dietary lactoferrin, few mechanisms from in vitro studies have been proposed to explain these properties. The best characterized lactoferrin targets are negatively charged molecules. They encompass pro-inflammatory microbial molecules, such as pathogen-associated molecular patterns (eg, lipopolysaccharide), but also host components such as DNA, the glycosaminoglycan chains of proteoglycans, and surface cell receptors. Signaling through these receptors is thought to be the main lever used by lactoferrin to influence immune cells and cytokine-balance-controlling cell activity. This article aims to review our current understanding, though incomplete, of the many ways lactoferrin influences the complex immune machinery and the known and putative mechanisms that may explain its properties.