Aging has a significant negative impact on human testicular function; steroidogenesis is gradually impaired, and testosterone replacement therapy still has many risks. Low-intensity pulsed ultrasound (LIPUS) has been used as a novel non-invasive treatment for male erectile dysfunction and other fields, and has been shown to increase testosterone levels in animal models. Testosterone is synthesized and secreted by Leydig cells (LCs), and the serum testosterone level decreases after aging due to the LCs senescence. However, the effect of LIPUS on human senescent LCs has not been reported. In this study, human senescent LCs were isolated and stimulated with different energy intensities in vitro, and cell morphology, cell apoptosis, cell proliferation, cell senescence levels, lipid droplet number, testosterone and INSL3 secretion levels were tested and analyzed. Quantitative Polymerase Chain Reaction (QPCR) and Western Blot were performed to compare cell senescence characteristics and the expression profile of key pathways of testosterone secretion, and transcriptome analysis was performed to explore the signaling pathways of LCs alteration after LIPUS stimulation. It was safe and effective to stimulate LCs with the 75 mW/cm² energy of LIPUS in vitro, which not only improved the senescence phenotype, but also effectively enhanced the secretory function of LCs in vitro, and increased the expression of key pathways of the testosterone synthesis pathway. These results suggest that LIPUS could be used as a novel treatment to human senescent LCs with decreased testosterone secretion levels in vitro.