Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.