The elimination process of systemically administered small interfering RNA (siRNA) was investigated by using siRNA labeled with an infrared fluorescent dye. A novel siRNA elimination pathway was identified. In this pathway, liver-enriched siRNA is secreted into the gallbladder and then emptied into the intestine. Blocking this pathway resulted in the absence of siRNA fluorescence within the intestine, with greatly enhanced siRNA accumulation in liver and gallbladder at the same time. Furthermore, we demonstrated that delivery carriers play an essential role in siRNA distribution and elimination, highlighting their importance in siRNA therapeutics.