Recent single-cell RNA sequencing of mouse XX and XY gonads across gonadal sex determination identified a rare cell population, designated as supporting-like cells (SLCs). SLCs primarily constitute the epithelium of the rete testis and ovarii in XY and XX gonads, respectively. The rete testis serves as an anatomical bridge between the testis and the efferent ducts, facilitating spermatozoa transport to the epididymis. The rete testis is composed of two distinct portions: an intratesticular and an extratesticular one. In the embryonic and neonatal testis, SLCs form the epithelium from the extratesticular rete testis to the straight tubules. SLCs represent the sole gonadal cell population expressing the transcription factor Pax8. The present study sought to elucidate the putative functions of rete testis epithelial cells in adult mice. This involved the utilisation of a genetic cell ablation technique based on differential sensitivity to diphtheria toxin, coupled with exploitation of the Pax8-specific expression in SLCs. It was found that only the intragonadal SLCs were able to undergo cell ablation efficiently. The ablation of intragonadal SLCs in mice results in efferent duct ligation-like effects, suggesting that these cells may be involved in the reabsorption of testis fluid.