Prevention of adhesion after tendon repair surgery is of considerable importance during tendon healing; however, current clinical outcomes are still not fully satisfactory. In this study, thermosensitive 5fluorouracil (5-Fu) loaded PLGA-PEG-PLGA hydrogels were used as injectable physical barriers for the prevention of tissue adhesion during tendon healing and inhibition of fibroblast proliferation. The 5-Fuloaded hydrogels showed a sol-gel-precipitation phase transition with increasing temperature, and the hydrogels displayed the maximum storage moduli at around physiological temperature. The sustained release of 5-Fu from the hydrogels lasted over 7 days. The PLGA-PEG-PLGA hydrogels degraded within 4 weeks after subcutaneous injection into rats, and showed acceptable biocompatibility in vivo. The antiadhesion efficacy of the hydrogels, with or without 5-Fu, during the Achilles tendon healing of rats was evaluated by macroscopic and histological analysis. It was found that the group treated with 5-Fuloaded hydrogels showed a significant inhibition of adhesion formation when compared to the untreated group or the group treated with the hydrogels only. Therefore, the 5-Fu-loaded injectable hydrogels hold potential as efficient physical barriers for the prevention of adhesion formation during Achilles tendon healing.