Phosphatidylinositol 5-phosphate (PtdIns5P), the least characterized among the three phosphatidylinositol monophosphates, is emerging as a bioactive lipid involved in the control of several cellular functions. Similar to PtdIns3P, it is present in low amounts in mammalian cells, and can be detected at the plasma membrane and endomembranes as well as in the nucleus. Changes in PtdIns5P levels are observed in mammalian cells following specific stimuli or stresses, and in human diseases. Recently, the contribution of several enzymes such as PIKfyve, myotubularins, and type II PtdInsP-kinases to PtdIns5P metabolism has gained a strong experimental support. Here, we provide a picture emerging from recent studies showing how this lipid can be generated and act as a regulator of membrane and cytoskeleton dynamics, and as a modulator of gene expression. We briefly summarize the current methods and tools for studying PtdIns5P, and discuss how PtdIns5P can integrate and coordinate different functions in a spatiotemporal manner.