The aim of this study was to investigate the role of an antichondrogenic factor, MIR221 (miR-221), in intervertebral disc degeneration (IDD), and provide basic information for the development of a therapeutic strategy for the disc repair based on specific nucleic acid based drugs, such as miR-221 silencing. We established a relatively quick protocol to minimize artifacts from extended in vitro culture, without selecting the different types of cells from intervertebral disc (IVD) or completely disrupting extracellular matrix (ECM), but by using the whole cell population with a part of resident ECM. During the de-differentiation process miR-221 expression significantly increased. We demonstrated the effectiveness of miR-221 silencing in driving the cells towards chondrogenic lineage. AntagomiR-221 treated cells showed in fact a significant increase of expression of typical chondrogenic markers including COL2A1, ACAN and SOX9, whose loss is associated with IDD. Moreover, antagomiR-221 treatment restored FOXO3 expression and increased TRPS1 expression levels attenuating the severity grade of degeneration, and demonstrating in a context of tissue degeneration and inflammation not investigated before, that FOXO3 is target of miR-221. Data of present study are promising in the definition of new molecules useful as potential intradiscal injectable biological agents.