Lamin proteins are essential structural elements of the nuclear envelope, critically involved in maintaining nuclear shape and mechanical stability. Lamin A/C, specifically, acts as a mechanotransducer that senses extracellular mechanical cues and transmits them into intracellular biochemical signals, thereby influencing cell adhesion, motility, and differentiation. Although microRNAs (miRNAs) have emerged as key regulators of cellular mechanotransduction pathways, the precise roles of miRNAs in modulating lamin A/C at the single-cell level remain poorly understood. Here, we utilized advanced biosensors based on fluorescence resonance energy transfer (FRET) and traction force microscopy to elucidate the impact of miRNA-548t-3p-induced lamin A/C downregulation on nuclear mechanical properties in single cells. Our findings demonstrate that miRNA-548t-3p specifically reduces lamin A/C levels, resulting in decreased nuclear tension and compromised focal adhesion dynamics. Furthermore, miRNA-548t-3p significantly diminishes the ability of cells to sense and respond to variations in extracellular matrix stiffness, leading to reduced cellular traction forces. These results underscore the pivotal role of lamin A/C in cellular mechanosensitivity and highlight miRNA-548t-3p as a critical modulator of nuclear mechanotransduction and mechanical homeostasis at the single-cell level. This study provides new insights into the complex interplay between miRNAs, nuclear mechanics, and cell-environment interactions, suggesting potential avenues for therapeutic intervention in diseases associated with disrupted mechanotransduction.