The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.