Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However， cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover， very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand， polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EGGlu)-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts， N-P(EGGlu)-IFN， C-IFN-P(EGGlu)， and C-IFN-PEG. Our results found circ-P(EGGlu)-IFN to show considerably greater stability， binding affinity， and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly， circ-P(EGGlu)-IFN exhibited longer circulation half-life， remarkably higher tumor retention， and deeper tumor penetration in vivo. As a result， administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together， our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.