Four-and-a-half LIM domains protein 2 (FHL2) is involved in major cellular mechanisms such as regulation of gene transcription and cytoskeleton modulation, participating in physiological control of cardiogenesis and osteogenesis. Knowledge on underlying mechanisms is, however, limited. We present here new data on FHL2 protein and its role during vertebrate development using a marine teleost fish, the gilthead seabream (Sparus aurata L.). In silico comparison of vertebrate protein sequences and prediction of LIM domain three-dimensional structure revealed a high degree of conservation, suggesting a conserved function throughout evolution. Determination of sites and levels of FHL2 gene expression in seabream indicated a central role for FHL2 in the development of heart and craniofacial musculature, and a potential role in tissue calcification. Our data confirmed the key role of FHL2 protein during vertebrate development and gave new insights into its particular involvement in craniofacial muscle development and specificity for slow fibers.