Profilins are small actin-binding proteins expressed in all eukaryotes. They are involved in the regulation of actin filament dynamics and various signalling pathways. The identification of a variety of profilin isoforms led to the assumption that there may be isoform-specific functions. In mammals, profilin-1 (PFN1) is ubiquitously expressed and engaged in the regulation of various motility processes in all cell types. By contrast, profilin-2a (PFN2a) is mainly restricted to neuronal cells and there is evidence that it is involved in neuronal plasticity and membrane trafficking. However, the PFN2a sequence is much better conserved than PFN1 throughout different phyla, indicating that its restricted expression and specialized function in mammals might be unique. Using isoform-specific antibodies, we show that the situation is different in birds. PFN2a is ubiquitously expressed in embryonic and adult chicken tissues at equal and frequently higher amounts than in mammals. Together with PFN1, it is present in cultivated chicken fibroblasts, but differentially localized. Knockdown experiments with miRNA reveal that PFN2a is involved in cell adhesion, spreading and locomotion, and silencing this isoform has pronounced consequences on these processes. Our results indicate profilin isoform expression is differentially regulated among vertebrates.