C4F7N (fluorinated nitrile) gas mixture has been utilized as the gas insulating medium to replace the most greenhouse gas SF6 (sulphur hexafluoride). Nowadays, there are few reports on the toxicity mechanism of C4F7N and studies on the application risk of C4F7N is insufficient. In this paper, we carried out acute toxicity tests for C4F7N gas systematically. The changes of vital signs of rats after exposure to C4F7N were analyzed and the influence of C4F7N on the main organs of rats was revealed for the first time. It was found that rats developed symptoms of respiratory rate decrease, respiratory mucosa damage, movement systems impairment and abnormal blood cell count after exposure to C4F7N. Pathological section results showed that 1.5% C4F7N could damage the lung, kidney, intestine and brain tissues of rats to a certain extent, but has little influence to the eye, skin, heart and liver. The LC50 (rat, 4 h) of C4F7N gas is in the range of 15,000 ppm (1.5%) and 20,000 ppm (2%). Relevant research results not only reveal the acute toxicity mechanism of C4F7N, but also provide important reference for the safety protection of scientific researcher, equipment production, engineering operation and maintenance personnel.