Cell migration is a fundamental process pertaining to many critical physiological events. The ability to form and release adhesion structures is necessary for cell migration. The Calpain family of cysteine proteases are known to target adhesion proteins as their substrates and modulate adhesion dynamics. The two best studied Calpains, Calpain 1 and Calpain 2 form catalytically active holoenzymes through heterodimerization with a common non-catalytic regulatory small subunit known as Calpain 4. In previous studies, we determined that calpains are important in the production of traction forces and in the sensing of localized mechanical stimulation from the external environment. We found that perturbation of either Calpain 1 or 2 had no effect on the generation of traction forces. However, traction forces were weak when Calpain 4 was silenced. On the other hand, silencing of Calpain 1, 2, or 4 resulted in deficient sensing of external mechanical stimuli. These results together suggest that Calpain 4 functions independent of the catalytic large subunits in the generation of traction forces but functions together with either catalytic subunit in sensing external mechanical stimuli. The small subunit Calpain 4 contains 268 a.a. and is composed of 2 domains, the N-terminal domain V and C-terminal domain VI. Domain VI is a calmodulinlike domain containing five consecutive EF-hand motifs, of which the fifth one heterodimerizes with a large subunit. Moreover, domain V contains the common sequence GTAMRILGGVI that suggests cell membrane interactions. Given these attributes of domain V and VI of Calpain 4, we speculated that an individual domain might provide the functional properties for either traction or sensing. Therefore, each domain was cloned and expressed individually in Capn4-/- cells and assayed for traction and sensing. Results revealed that over-expression of domain V was sufficient to rescue the traction forces defect in Capn4-/- cells while overexpression of domain VI did not rescue the traction force. Consistent with our hypothesis, overexpression of domain VI rescued the sensing defect in Capn4-/- cells while overexpression of domain V had no effect. These results suggest that individual domains of Calpain 4 do indeed function independently to regulate either traction force or the sensing of external stimuli. We speculate that membrane association of Calpain 4 is required for the regulation of traction force and its association with a catalytic subunit is necessary for mechanosensing.