Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1(- / - ) mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.