Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are defining components of farmed fish feeds. They are known effectors of bone metabolism and may reduce the occurrence of skeleton deformities in farmed fish, given that dietary levels are optimized. Underlying mechanisms of bone-related effects of PUFAs are however poorly understood. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of AA, EPA and DHA on cell proliferation, extracellular matrix (ECM) mineralization and expression of a selection of marker genes. While all fatty acids promoted morphological changes and stimulated cell proliferation, AA and EPA inhibited ECM mineralization and DHA had the opposite effect. When fatty acids were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. qPCR data indicated that, while expression of COL1A1, ON, MGP and RUNX2 genes remained unaltered, that of OP, BMP2, TNAP and COX2 genes was significantly regulated upon exposure to individual PUFAs. The concomitant increase in BMP2 and TNAP gene expression in DHA-treated cells and the strong decrease in OP transcript in AA- and EPA-treated cells may explain the pro- and anti-mineralogenic effects of these fatty acids, respectively. An increase in COX2 gene expression was also observed in EPA- and DHA-treated cells but the biological significance of this result remains ambiguous. This work also demonstrated the suitability of our in vitro cell systems to get insights into bone-related effects of PUFAs and to investigate the intracellular pathways involved in their proliferative and mineralogenic effects.