Abstract Background We previously reported that membrane-type 5-matrix metalloproteinase (MT5-MMP) deficiency not only reduces pathological hallmarks of Alzheimer’s disease (AD) in 5xFAD (Tg) mice in vivo, but also impairs interleukin-1 beta (IL-1β)-mediated neuroinflammation and Ab production in primary Tg immature neural cell cultures after 11 days in vitro. We now investigated the effect of MT5-MMP on incipient pathogenic pathways that are activated in cortical primary cultures at 21-24 days in vitro (DIV), during which neurons are organized into a functional mature network. Methods Using wild-type (WT), MT5-MMP -/- (MT5 -/- ), 5xFAD (Tg) and 5xFADxMT5-MMP -/- (TgMT5 -/- ) mice, we generated primary neuronal cultures that were exposed to IL-1b and/or different proteolytic system inhibitors. We assessed neuroinflammation, APP metabolism, synaptic integrity and electrophysiological properties using biochemical, imaging and whole- cell patch-clamp approaches. Results The absence of MT5-MMP impaired the IL-1b-mediated induction of inflammatory genes in TgMT5 -/- cells compared to Tg cells. Furthermore, the reduced density of dendritic spines in Tg neurons was also prevented in TgMT5 -/- neurons. IL-1b caused a strong decrease in the dendritic spine density of WT neurons, which was prevented in MT5 -/- neurons. However, the latter exhibited fewer spines than the WT under untreated conditions. The spontaneous rhythmic firing frequency of the network was increased in MT5 -/- neurons, but not in TgMT5 -/- neurons and IL-1b increased this parameter only in Tg neurons. In terms of induced somatic excitability, Tg and TgMT5 -/- neurons exhibited lower excitability than WT and MT5 -/- , while IL-1b impaired excitability only on non-AD backgrounds. The synaptic strength of miniature global synaptic currents was equivalent in all genotypes, but increased dramatically in WT and MT5 -/- neurons after IL-1b. MT5-MMP deficiency decreased endogenous and overexpressed C83 and C89 levels but did not affect Ab levels. C99 appears to be cleared by several pathways, including g-secretase, the autophagolysosomal system and also a-secretase, via its conversion to C83. Conclusion In summary, this study confirms that MT5-MMP is a pivotal factor affecting not only neuroinflammation and APP metabolism but also synaptogenesis and synaptic activity at early stages of the pathology, and reinforces the relevance of targeting MT5-MMP to fight AD.