Tendon remodeling relies on extracellular matrix (ECM) restructuring by the matrix metallopeptidases (MMPs). The aim of this study was to investigate MMP-2 activity in different regions of the calcaneal tendon (CT) after resistance training (RT) in ovariectomized rats. Wistar adult female rats were grouped into sedentary (Sed-Intact), ovariectomized sedentary (Sed-Ovx), acute exercise (AcuteEx-Intact), ovariectomized acute exercise (AcuteEx-Ovx), resistance trained (ChronicEx-Intact), and ovariectomized resistance trained (ChronicEx-Ovx) (n = 10 each group). The RT protocol required the animals to climb a 1.1-m vertical ladder with weights attached to their tail. The sessions were performed once every 3 days with 4-9 climbs and 8-12 dynamic movements per scaling. The acute groups performed one session and the chronic groups underwent 12 weeks of RT. There was an increase in total MMP-2 activity in Sed-Ovx, AcuteEx-Intact, and ChronicEx-Intact compared with that in Sed-Intact in the proximal region of CT. AcuteEx-Ovx exhibited higher total MMP-2 than Sed-Ovx and AcuteEx-Intact in the distal region of CT. Chronic-Ovx presented lower total MMP-2 activity than Sed-Ovx and Chronic-Intact in the distal region of tendon. The active MMP-2 was higher for the AcuteEx-Ovx than Sed-Ovx and AcuteEx-Intact in proximal region of tendon. There was higher active MMP-2 in the distal region of tendon in the Acute-Ovx than in the Sed-Ovx and AcuteEx-Intact. Ovariectomy and resistance exercise modulate MMP-2 activity according to specific tendon region, indicating a differentiated tissue remodeling.