Summary Background Skin health declines with age and this is partially attributed to immunosenescence. Mast cells (MCs) are innate immune cells that coordinate tissue immune responses integral to skin homeostasis and disease. Objectives To understand how MCs contribute to human skin ageing, we investigated how intrinsic ageing impacts MC phenotype and MC relationships with other immune cells and skin structures. Methods In photoprotected skin biopsies from young (≤ 30 years) and aged (≥ 75 years) individuals, immunostaining and spatial morphometry were performed to identify changes in MC phenotype, number, distribution and interaction with the vasculature and nerve fibres. Quantitative polymerase chain reaction was used to measure changes in gene expression related to immune cell activity and neuropeptide signalling. Results Skin MCs, macrophages and CD8+ T cells increased in number in intrinsically aged vs. young skin by 40%, 44% and 90%, respectively (P < 0·05), while CD4+ T cells and neutrophils were unchanged. In aged skin, MCs were more numerous in the papillary dermis and showed a reduced incidence of degranulation (50% lower than in young, P < 0·01), a conserved tryptase–chymase phenotype and coexpression of granzyme B. In aged skin, MCs increased their association with macrophages (~ 48% vs. ~27%, P < 0·05) and nerve fibres (~29% vs. 16%, P < 0·001), while reducing their interactions with blood vessels (~34% vs. 45%, P < 0·001). Additionally, we observed modulation of gene expression of vasoactive intestinal peptide (VIP; increased) and substance P (decreased) with age; this was associated with an increased frequency of VIP+ nerve fibres (around three times higher in aged skin, P < 0·05), which were strongly associated with MCs (~19% in aged vs. 8% in young, P < 0·05). Conclusions In photoprotected skin we observed an accumulation of MCs with increasing age. These MCs have both altered functionality and distribution within the skin, which supports a role for these cells in altered tissue homeostasis during ageing. What's already known about this topic? In aged skin, immunity becomes dysregulated leading to greater baseline inflammation and dampened adaptive immunity. Mast cells (MCs) are regarded as multifunctional regulators of tissue homeostasis and immunity and are known to increase in number in the skin with age. What does this study add? This study shows that the increase in MCs in aged skin is localized to the papillary dermis, where these cells are in closer proximity to macrophages but have reduced interaction with the microvasculature and other immune populations. We show that in aged skin, MCs also exhibit lower amounts of degranulation and form closer interactions with macrophages and vasoactive intestinal peptide‐positive nerve fibres while lessening their association with the dermal vasculature. What is the translational message? Alterations in MC frequency, functionality and distribution may contribute to the immunosenescent skin phenotype associated with intrinsic ageing. The changes in MC behaviour may also play a role in neurogenic inflammation and could provide a therapeutic target for age‐associated pruritus. Respond to this article