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Metabolites are critical products and mediators of cellular and tissue function, and key signals in cell-to-cell, organ-to-organ and cross-organism communication. Many of these interactions are spatially segregated. Thus, spatial metabolomics can provide valuable insight into healthy tissue function and disease pathogenesis. Here, we review major mass spectrometry-based spatial metabolomics techniques and the biological insights they have enabled, with a focus on brain and microbiota function and on cancer, neurological diseases and infectious diseases. These techniques also present significant translational utility, for example in cancer diagnosis, and for drug development. However, spatial mass spectrometry techniques still encounter significant challenges, including artifactual features, metabolite annotation, open data, and ethical considerations. Addressing these issues represent the future challenges in this field.