To investigate the mechanism for the formation of tubule-like structures in testicular organoids, we conducted proteomic analysis on O-Torgs and T-Torgs at day 6. Through differential protein expression and enrichment analysis, it was found that the extracellular matrix (ECM) related pathways in O-Orgs were significantly upregulated. This indicates that the ECM might facilitate the formation of tubule-like structures in O-Orgs, which might be important for spermatogenesis.