The capability to engraft into degenerative environment or damaged tissues is considered crucial to maximize the therapeutic effect of stem cells in tissue regeneration. Human chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from the placenta have been reported to have therapeutic effects in animal models of liver injury. However, the effect of transplantation route has not been evaluated. Thus, we investigated to identify optimal transplantation conditions of CP-MSCs for functional recovery of injured liver. PKH26 labeled CP-MSCs was engrafted into carbon tetrachloride (CCl4)-injured rat model through direct transplantation into the liver (DTP), intrasplenic transplantation (STP), and intravenous transplantation via the tail vein (TTP). Non transplanted (NTP) rats were maintained as sham controls. Serum and liver tissues were analyzed post 1-, 2-, 3-week after transplantation. The engraftment of cells was higher in DTP and STP group until 3-week post transplantation. In blood chemistry, the levels of glutamate-oxaloacetate transaminase (GOT/AST), glutamate-pyruvate transaminase (GPT/ALT) and total bilirubin (TBIL) in DTP and STP rats were significantly decreased compare to those of NTP rats (p < 0.01). In addition, the expression and deposition of type I collagen in DTP and STP rats was significantly reduced when they compared with NTP animals (p < 0.01). Therapeutic efficacy was better in DTP and STP rats than in TTP group. These results suggest that the administration of CP-MSCs via STP is an effective way for their therapeutic potential. These results provide useful guidelines for the application of basic transplantation technology to the cell therapy of liver disease using placenta-derived stem cells.