Stromal vascular fraction (SVF), an uncultured adipose-derived cell population, exerts regenerative effects primarily through paracrine mechanisms. While local intratesticular injection of SVF alleviates busulfan-induced testicular injury, the intravenous route remains unexplored. This study investigated whether intravenous SVF administration could mitigate busulfan-induced spermatogenic impairment. Adult male Sprague-Dawley rats received busulfan (15 mg/kg, intraperitoneal) or vehicle; the busulfan + SVF group received 2 × 10⁶ allogeneic SVF cells intravenously immediately after busulfan injection. Two weeks later, we evaluated sperm parameters, testicular histopathology (Johnsen’s score, seminiferous tubule diameter, germinal epithelial thickness), and immunohistochemical expression of SOX9 (Sertoli cells), CYP11A1 (Leydig cells), and DDX4 (germ cells). Busulfan alone induced severe spermatogenic defects, including reduced sperm count and motility, increased abnormal morphology, marked histopathological damage, and decreased expression of SOX9, CYP11A1, and DDX4. In contrast, intravenous SVF treatment significantly improved sperm count, motility, and testicular architecture, and restored the expression of SOX9, CYP11A1, and DDX4 within two weeks. This is the first demonstration that intravenous SVF attenuates early busulfan-induced testicular injury, providing preliminary evidence for a rapid, culture-free approach as a less invasive alternative to local injection. However, longer follow-up and fertility studies are needed to confirm sustained spermatogenic restoration.