Objective: The study aim was to prepare poly-DL-lactide-poly (PELA) microspheres encapsulating recombinant tissue inhibitors of metalloproteinase-1 (TIMP-1) in an adenovirus to investigate its inhibition on the proliferation of hepatocellular carcinoma cells HepG2. Methods: Microspheres were prepared by encapsulating the recombinant TIMP-1 adenovirus into biodegradable PELA. The particle size, viral load, encapsulation efficiency and in-vitro release were measured. Microspheres were used to infect HepG2 cells, then infection efficiency was examined under a fluorescent microscope and ultrastructural changes assessed by TEM. Expression of TIMP-1 mRNA in HepG2 cells was examined by semi-quantitative RT-PCR and proliferation by MTT and cell growth curve assays. Results: We successfully prepared microspheres encapsulating recombinant TIMP-1 adenovirus with a diameter of $1.965{mu}m$, an encapsulation efficiency of 60.0%, a viral load of $10.5{ imes}10^8/mg$ and approximate 60% of virus release within 120 h, the total releasing time of which was longer than 240 h. The microspheres were confirmed to be non-toxic with blank microspheres. Infected HepG2 cells could stably maintain in-vitro expression of TIMP-1, with significantly effects on biological behaviour Conclusion: PELA microspheres encapsulating a recombinant TIMP-1 adenovirus can markedly inhibit the proliferation of HepG2 cells, which provides an experimental basis for polymer/chemistry-based gene therapy of hepatocellular carcinomas.