It took over seven hours but a team of 30 doctors, which included 18 surgeons from different specialties at the Mayo Clinic, to complete the surgery.
The team untangled the livers, repositioned the hearts and divided a shared intestine in the twins who were born five months ago joined at the chest and abdomen.
The twins Abbigail and Isabelle Carlsen born to a North Dakota couple, are said to be recovering well but remain in intensive care under sedation, and are breathing with the help of ventilators.
The twins had spent the first five months of their life eye to eye.
Their condition is described as critical but their vital signs are stable.
Doctors will closely monitor their conditions as the first 24 to 48 hours is believed to be critical.
The twins have been under the care of the Mayo Clinic team since February 24, who studied their anatomy and prepared them for the surgery.
The separation surgery was a series of one major surgery after another.
The first step in the procedure separated the chest walls and their hearts were then re-positioned.
The twins' gall bladders were then removed and later re-routed as planned.
The Mayo clinic surgeons then began the liver separation, which included the meticulous identification of vascular and biliary structures of the liver.
The twins' livers were separated, as were their pancreases and all other major organs.
For Isabelle who retained the common bile duct, a bowel was reconstructed and Abbigail also went through bowel and biliary reconstruction.