The small heat shock protein Hsp27 or its murine homologue Hsp25 acts as an ATP-independent chaperone in protein folding, but is also implicated in architecture of the cytoskeleton, cell migration, metabolism, cell survival, growth/differentiation, mRNA stabilization, and tumor progression. A variety of stimuli induce phosphorylation of serine residues 15, 78, and 82 in Hsp27 and serines 15 and 86 in Hsp25. This post-translational modification affects some of the cellular functions of Hsp25/27. As a consequence of the functional importance of Hsp25/27 phosphorylation, aberrant Hsp27 phosphorylation has been linked to several clinical conditions. This review focuses on the different Hsp25/27 kinases and phosphatases that regulate the phosphorylation pattern of Hsp25/27, and discusses the recent findings of the biological implications of these phosphorylation events in physiological and pathological processes. Novel therapeutic strategies aimed at restoring anomalous Hsp27 phosphorylation in human diseases will be presented.