In this work we have studied the influence of phosphorylation of the Sgt1 protein (a Hsp90 co-chaperone) on its nuclear translocation. For that we have prepared non-phosphorylable Sgt1 mutants such as S249A, S299A, S249/299A or a phosphorylation mimic S299D mutant and we have found that, after heat shock the mutants representing the non-phosphorylated forms of Sgt1 translocate to the nucleus more efficiently than wild type Sgt1 and the S299D mutant. Moreover, we have shown that S100A6, a calcium binding protein, is required for Sgt1 translocation and that upon heat shock S100A6 translocates to the nucleus together with Sgt1. We have also found that the non-phosphorylable S299A Sgt1 mutant interacts more efficiently with S100A6 and less efficiently with Hsp90 than wild type Sgt1 which suggests that dephosphorylation of Sgt1 decreases the Sgt1-Hsp90 complex stability. In conclusion, in this work, we have elucidated for the first time the phosphorylation-dependent mechanism of Sgt1 nuclear translocation.