For a valid cytokine immunotherapy of malignancies, a suitable delivery system that ensures slow-release of cytokines is required, because short half-life in vivo of the molecules ruins therapeutic efficacy while causing severe systemic toxic effects. We previously showed that the cholesterol-bearing pullulan (CHP)-based hydrogel nanoparticles, or nanogel, encapsulates, stabilizes and releases various molecules. Here we applied this nanogel to administration in vivo of interleukin-12 (IL-12). Recombinant murine IL-12 (rmIL-12) was successfully incorporated into CHP nanogel simply by incubated with CHP at room temperature. After subcutaneously injected into mice, the CHP/rmIL-12 complex led to a prolonged elevation in IL-12 concentration in the sera. Repetitive administrations of the CHP/rmIL-12, but not rmIL-12 alone, induced drastic growth retardation of preestablished subcutaneous fibrosarcoma without causing any serious toxic event. The present study proposes a novel therapeutic intervention technology, taking advantage of slow and sustained release of bioactive cytokines from the self-assembling biocompatible nanoparticles.