A single incision laparoscopic system (SILS) was used to remove the falciform ligament of an adult male cheetah (Acinonyx jubatus) during routine diagnostic liver biopsy. Adipose tissue isolated from the falciform ligament was used to establish a mesenchymal stem cell culture. The use of a SILS port for liver biopsy and falciform ligament collection allowed for a large amount of fat to be collected from a small surgical incision and rapid postoperative recovery. This case expands the use of the single incision laparoscopy surgical technique beyond reproductive sterilization procedures in large cats.