S100 protein family has been implicated in multiple stages of tumorigenesis and progression. Among the S100 genes, 22 are clustered at chromosome locus 1q21, a region frequently rearranged in cancers. S100 protein possesses a wide range of intracellular and extracellular functions such as regulation of calcium homeostasis, cell proliferation, apoptosis, cell invasion and motility, cytoskeleton interactions, protein phosphorylation, regulation of transcriptional factors, autoimmunity, chemotaxis, inflammation and pluripotency. Many lines of evidence suggest that altered expression of S100 proteins was associated with tumor progression and prognosis. Therefore, S100 proteins might also represent potential tumor biomarkers and therapeutic targets. In this review, we summarize the evidence connecting S100 protein family and cancer and discuss the mechanisms by which S100 exerts its diverse functions.