An amphiphilic gelatin macromolecule capable of self-assembling to form micelle-like nanospheres for entrapping hydrophobic therapeutic molecules was successfully synthesized by grafting hydrophobic hexanoyl anhydrides to the amino groups of primitive gelatin. The substitution and grafting efficiency of the modified gelatin displayed a concentration-dependent manner toward the hexanoyl anhydride. The particle size and stability of the resulting self-assembled nanoparticles strongly depend on the degree of substitution of the gelatin. These gelatin nanoparticles exhibited an outstanding cytocompatibility and highly efficient drug encapsulation efficiency. The drug release profile of the anticancer agent (camptothecin) from the amphiphilic gelatin nanoparticles shows that the drug release mechanism is dominated by a combined effect of diffusion and degradation. Furthermore, a tunable sustained release and degradation behavior can be controlled by changing the degree of substitution of the gelatin. This new type of amphiphilic gelatin nanoparticles with controlled biodegradability and rapid cellular internalization may serve in the intracellular delivery of specific proteins or genes, permitting a novel design for intracellularly-based nanotherapy.