Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor germ cells-derived sperm production. This technique has the potential to be used as an alternative strategy for producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. The objective of the present study was to investigate whether the homologous transplantation of isolated buffalo germ cells could result in colonization of recipient testes. Germ cells were isolated from prepubertal buffalo testes (4–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin lectin-coated dishes and after that Percoll density gradient centrifugation as descrived by van Pelt et al. (1996) with minor modifications. A discontinuous density gradient was prepared with 60, 50, 40, 36, 34, 32, 30, 28, and 20% Percoll in a 15-mL centrifuge tube. The enriched germ cells were then labelled with red fluorescent linker dye PKH26 (Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions, and ~10 million cells/testis were transferred into the rete testis of 3 recipients (16–18 months of age) under ultrasonographic guidance. After 45 days, testes were surgically removed and samples were prepared for analysis of labelled cells via wet mount of seminiferous tubules and individual cells isolation. When wet mount specimen were observed under a fluorescence microscope, PKH26-positive cells were identified on the seminiferous tubule basement membrane in all 3 recipients, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be donor germ cells. In freshly isolated cells, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the seminiferous tubules. In conclusion, we report successful homologous transplantation of germ cells in prepubertal buffalo testes. Further studies will investigate functionality of transferred testicular cells.