Fibroblasts can be transformed into myofibroblasts under pro-fibrotic conditions, which is characterized by increased contractility and reduced matrix degradation. The relationship between contractile activity and matrix degradation is not fully understood. We found that TGF-β1-induced myofibroblast activation occurs on a culture dish, favoring stress fiber formation and inhibiting podosome structures due to high matrix stiffness. To mimic physiological conditions, we cultured fibroblasts on collagen gel. Blocking actomyosin signaling significantly reduced TGF-β1-induced myofibroblast activation. Tpm1.6, an actomyosin-associated contractile unit, was specifically upregulated by TGF-β1 on soft collagen substrates. Depletion of Tpm1.6 attenuated TGF-β1-induced increase of α-SMA, N-cadherin, and β1-integrin, indicating its crucial role in early myofibroblast activation during fibrosis progression. Tpm1.6 depletion reduced TGF-β1-induced cell contractility and enhanced collagen degradation. Notably, in Tpm1.6-depleted fibroblasts, TGF-β1 triggered formation of distinct α-SMA dot structures enriched with MMP9, promoting collagen degradation. Our study highlights the pivotal role of Tpm1.6 in TGF-β1-induced myofibroblast activation and collagen degradation. Depletion of Tpm1.6 triggers robust collagen degradation through distinct α-SMA dots, presenting a potential therapeutic approach for chronic kidney disease.