This study aimed to evaluate the expression of calpastatin (CAST) isoforms and their potential associations with fiber type composition (%RA), calpastatin activity (CA) and myofibril fragmentation index (MFI) in three muscles with known differences in tenderness (infraspinatus, triceps brachii and semitendinosus) of Angus steers. Expression of total CAST (CAST-T) and CAST isoforms I, II, III (2–3) and III (2–4) (including or not exon 3) was evaluated by qRT-PCR. CAST expression and CA were significantly higher and MFI was lower in semitendinosus, the muscle with the highest %RA of IIX fibers. Differential expression of isoforms defined the variability in CAST-T among muscles. Semitendinosus had a higher expression of isoforms II and III (2–3), but lower expression of III (2–4) compared to the other two muscles. Relative expression of isoforms II and III that were defined by promoter preference linked to alternative splicing, seem to be the main factors explaining differences in CAST expression and ultimately in MFI among muscles.