The nucleolus is a subnuclear compartment with key roles in rRNA synthesis and ribosome biogenesis, complex processes that require hundreds of proteins and factors. Alterations in nucleolar morphology and protein content have been linked to the control of cell proliferation and stress responses and, recently, further implicated in cell senescence and ageing. In this study, we report the functional role of NOL12 in the nucleolar homeostasis of human primary fibroblasts. NOL12 repression induces specific changes in nucleolar morphology, with increased nucleolar area but reduced nucleolar number, along with nucleolar accumulation and increased levels of fibrillarin and nucleolin. Moreover, NOL12 repression leads to stabilization and activation of p53 in an RPL11-dependent manner, which arrests cells at G2 phase and ultimately leads to senescence. Importantly, we found NOL12 repression in association with nucleolar stress-like responses in human fibroblasts from elderly donors, disclosing it as a biomarker in human chronological aging.