Nivolumab is an anti-PD1 antibody approved for the treatment of metastatic melanoma (MM), for which still limited evidence is available on the correlation between drug exposure and patient’s outcomes. In this observational retrospective study we assessed whether nivolumab concentration is associated with treatment response in patients with MM, and if patient’s genetic profile plays a role in this association. We observed a statistically significant correlation between nivolumab serum concentration and clinical outcomes, measured as overall survival and progression free survival. Moreover, patients who reached a clinical response (CR) had significantly higher concentration of nivolumab and presented a distinct genetic signature, with a more marked activation of ICOS and other genes involved in effector T-cells mediated pro-inflammatory pathways. In conclusion, in patients with MM nivolumab concentration correlates with clinical outcomes and is associated with an increased expression of ICOS and other genes involved in the activation of T effectors cells.