Sustained delivery of proteins from polymer-based thermosensitive gel has achieved considerable attention since last decade. In our previous work, we developed a formulation for sustained delivery of IL-1Ra-loaded poloxamer 407 formulation and investigated its in vitro and in vivo characteristics. In the present work, we extended this approach to investigate stability of IL-1Ra from poloxamer 407 formulation stored at 4 °C, 25 °C and 40 °C for 3 months. Samples were taken and in vitro drug release kinetics was studied. Percent of drug content was measured using the BCA method. DSC and SDS-PAGE were used to assess the conformational stability of IL-1Ra. FTIR spectroscopy was performed to investigate the drug-polymer interaction. From the results, it was found that gelation temperature, viscosity and in vitro release pattern of IL-1Ra from poloxamer 407 formulation at 4 °C were almost same throughout the stability study period. DSC profiles of IL-1Ra loaded in poloxamer 407 formulation increased the thermostability of IL-1Ra significantly in poloxamer 407 formulation. There were no apparent changes in the entire FTIR spectrum of the IL-1Ra that would suggest that there was no effect of the polymer on the structure of IL-1Ra. Moreover, results of SDS-PAGE confirmed the stability of IL-1Ra in poloxamer 407 formulation. These results provided evidence that poloxamer 407 is a promising polymer not only for sustained delivery of IL-1Ra but also provides conformational stability for extended time.