Harvesting human energy currently occurs to power wearable devices or monitor human signs, these applications make the energy harvester less applicable to and seldom used for assisting human motion. However, there is a high demand for using such collected energy with the assistance of human motion. This paper presents a novel energy harvester that is designed to collect negative work, assist human motion, and realize self-powering. An active self-powered human motion assist system (HMAS) is developed. The system consists of a human motion assist device, a flexible rack, an electronic circuit module, and a supercapacitor. The HMAS can collect negative work from the human body, provide the user with additional motion assistance, and reduce stamina consumption. A series of experiments verify that HMAS has a high negative work collection power and a high energy conversion efficiency. The average output power is 0.93 W measured by the negative work collection test bed at a simulated knee bend angle of 40° and a frequency of 2 Hz. The energy conversion efficiency is up to 48.2%. Human motion assistance experiments verify that HMAS can provide volunteers with up to 2.57% assisting moment and minimize the metabolic cost of volunteers by 6.07% compared to without wearing HMAS. This research work is proposed to contribute to the development of active self-powered exoskeleton technology. This technology can be practically applied in the fields of rehabilitation therapy, logistics transportation, and military combat.