Gardeniae Fructus is a traditional medicine used for the treatment of contusion such as ankle sprain. Geniposide is one of the main components of Gardeniae Fructus with diverse biological activities. In order to gain further insight into the therapeutic action of Gardeniae Fructus extract (GFE) and geniposide on ligament injuries, a new in vitro model was developed in the present study. Rat hind ankle ligament fibroblasts (RHALFs) derived from Sprague-Dawley rats were cultured, and the cell proliferation and collagen content were examined by MTT and a Sirius Red-based colorimetric assay after stimulating with each drug. The cell growth of RHALFs was promoted by culturing with 37.5-150 microg/mL of GFE and 25-200 microM of geniposide. The content of collagen in the RHALFs was significantly increased up to 131.4% and 124.2% of the control value by culturing with the GFE and geniposide, respectively. By contrast, both cell growth and collagen content were impaired by adding 25-200 microM of diclofenac, one of the common medications for ligament injuries. The findings suggest that GFE and geniposide may ameliorate the treatment of ligament injuries by proliferating ligament fibroblasts and promoting the synthesis of collagen. However, the use of diclofenac to treat acute ligament injuries should be reassessed although it possesses a potential effect on relieving symptoms.