Updated project metadata. Lipids play a crucial role in signalling and metabolism, regulating the development and maintenance of the skeleton. Membrane lipids have been hypothesised to act as intermediates upstream of orphan phosphatase 1 (PHOSPHO1), a major contributor to phosphate generation required for bone mineralisation. Here, we spatially resolve the lipid atlas of the healthy mouse knee and demonstrate the effects of PHOSPHO1 ablation on the growth plate lipidome. Lipids spanning 17 subclasses were mapped across the knee joints of healthy juvenile and adult mice using matrix-assisted laser desorption ionisation imaging mass spectrometry (MALDI-IMS), with annotation supported by shotgun lipidomics. Multivariate analysis identified 96 and 80 lipid ions with differential abundances across joint tissues in juvenile and adult mice respectively. In both ages, marrow was enriched in phospholipid platelet activating factors (PAFs) and related metabolites, cortical bone had a low lipid content, while lysophospholipids were strikingly enriched in the growth plate, an active site of mineralisation and PHOSPHO1 activity. Spatially-resolved profiling of PHOSPHO1-knockout (KO) mice across the resting, proliferating, and hypertrophic growth plate zones revealed 272, 306, and 296 significantly upregulated, and 155, 220 and 190 significantly downregulated features, respectively, relative to wild type (WT) controls. Of note, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine and phosphatidylethanolamine derived lipid ions were upregulated in PHOSPHO1-KO versus WT. Our imaging pipeline has established a spatially-resolved lipid signature of joint tissues and has demonstrated that PHOSPHO1 ablation significantly alters the growth plate lipidome, highlighting an essential role of the PHOSPHO1-mediated membrane phospholipid metabolism in lipid and bone homeostasis.