Megachile rotundata is a solitary cavity nesting bee that is the primary commercial pollinator of alfalfa in North America. During prepupal to adult development, they may be exposed to fungal, parasitic, and bacterial pathogens. However, little is known about their immune function throughout pupation. We characterized functional immunity of M. rotundata across development stages. We injected prepupal, pupal, and adult bees with live E. coli and compared mortality across groups. We also developed an assay to measure antimicrobial peptide (AMP) activity in hemolymph for the same age groups. Both pupal and prepupal bees are sensitive to injected E. coli, resulting in high mortality, while adult bees survived longer after infection. Pupal bees had significantly less AMP activity compared to prepupae and adults. Understanding immunity of M. rotundata will provide context for improving commercial rearing practices, where measuring AMP activity can now serve as a biological marker of bee quality.