Commercial live attenuated influenza vaccines (LAIVs) usually contain a high proportion of defective interfering particles (DIPs). Given that LAIVs are not sufficiently protective worldwide, the potential to enhance their efficacy by reducing the proportion of DIPs remains largely unknown. In this study, a prepared H3N2 cold-adapted LAIV with a low proportion of DIPs exhibits delayed yet improved replication in the upper respiratory tract of mice. The low DIPs LAIV induces an increase in goblet cells, microfold cells, and neutrophils, along with enhanced antigen presentation by dendritic cells. Compared to the commercially sourced high DIPs LAIV, the low DIPs LAIV elicits enhanced mucosal and humoral immune responses, facilitates cross-neutralization in mice, and provides complete protection against lethal challenges with H3N2, H1N1 or H1N1pdm09 strains. This study offers insights into optimizing commercial LAIVs and replicative RNA virus-based vaccines by controlling DIPs.