Background: Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA approved biologic drug for antagonizing IL-1 effects in patients with Rheumatoid arthritis. Notably, the less expensive production of this drug might help reduce the final therapeutic costs. Objectives: This study aimed to evaluate the possibility of producing biologically active recombinant IL-1Ra by a single step purification procedure mediated by a self-cleavable intein. Methods: Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) in fusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations, was performed on A375 and HEK293 cells treated by a constant concentration of IL-1β (2ng/mL). Results: IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1β was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which it was 96% for the inhibitory effects of the standard drug. Conclusion: In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable for producing other biologics.