MicroRNAs (miRs) play significant roles in diverse biological processes. Previous studies have primarily focused on the conventional RNA interference (RNAi) mechanism of miRs and have largely attributed their biological functions to RNAi-mediated gene regulation. However, our lab discovery of a novel, evolutionarily conserved biophysical action of miRs challenges this conventional view. We found that miR1 directly binds to ion channels and modulates cardiac electrophysiology through a biophysical mechanism. Our findings further demonstrate that the RNAi and biophysical functions of miR1 are distinct and independent mechanisms, and that selective disruption of miR1’s biophysical action leads to severe heart functional decline. These results suggest that the biophysical actions of miR1 may extend beyond ion-channel regulation and may broadly contribute to cardiac physiology. Based on this concept, we proposed an innovative hypothesis that miR1 physically interacts with myofilament regulatory protein(s) and biophysically modulate cardiac contractile force. Using crosslink RNA pulldown-mass spectrometry assays, we identify cardiac troponin proteins as candidate miR1-interacting proteins, whereas other major myofilament proteins were not detected. After validating their physical interactions, we used skinned rodent cardiac muscle preparations treated with or without extracellular microRNAs to determine whether miR1 directly regulates myofilament function. We found that treatment with miR1 or Mu14G caused a right shift of the Ca²⁺-force curve and decreased pCa50, indicating reduced myofilament Ca²⁺ sensitivity during muscle force production. Our investigation of miR1’s biophysical modulation of intracellular myofilament regulatory protein(s) in cardiomyocytes is groundbreaking. This work expands the current understanding of miR biology beyond canonical RNAi and opens promising therapeutic opportunities for dilated cardiomyopathy and other cardiac diseases through the development of RNA-based medicine.