Sustained muscle loss leads to weakening and severe metabolic consequences. Immobilization-induced muscle atrophy worsened in the tibialis anterior (TA) muscle immediately after cast removal and was associated with connective tissue alterations and sustained activation of proteolysis, suggesting a cross-talk between extracellular matrix (ECM) and muscle fibres. We investigated ECM alterations and integrin signalling in the immobilized and remobilized TA. Adult rats were subjected to unilateral hindlimb casting for 8 days (I8). Casts were removed at I8 and animals were allowed to recover for 10 days (R1 to R10). The immobilization-induced connective tissue thickening in the TA resulted from an increase in perimysium at I8 and in endomysium after remobilization. These alterations correlated with additional ramifications in the ECM during early recovery and with a decrease of muscle fibre area associated with their deformation. Levels of mRNA encoding two matricellular proteins, i.e. tenascin-C and Sparc, increased immediately after cast removal, while mRNA and/or protein levels ILK, Parvin-α, integrin-α7 and -β1 increased during immobilization and early recovery. Finally, mRNA levels for MyoD and Myf5 increased during immobilization and returned to basal levels at R6. In conclusion, the TA muscle atrophy during remobilization was associated with pronounced structural changes of the endomysium, suggesting changes in the transmission of contractile forces from adjacent muscle fibres within fascicles. In addition, the up-regulation of integrin scaffold proteins and of some matricellular proteins that regulate myogenesis during immobilization and remobilization suggests that integrin signalling was enhanced and may influence skeletal muscle recovery