Ca-regulation of cardiac contractility is mediated through the troponin complex， which comprises three subunits: cTnC， cTnI， and cTnT. As intracellular [Ca] increases， cTnI reduces its binding interactions with actin to primarily interact with cTnC， thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD)， the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD， we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1-167) and cTnI(1-193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers， and their influence on Ca-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9?μm) and long (2.2?μm) sarcomere lengths (SLs). Our results show that the cTnI(1-167) truncation diminished the SL-induced increase in Ca-sensitivity of contraction， but not the SL-dependent increase in maximal tension， suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT)， both truncations displayed greater Ca-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore， cTnI(1-167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.