SUMMARYThe present study aimed to explore the molecular factors underlying differences in Calpain/Calpastatin proteolytic system in Nellore and Angus cattle. Longissimus muscle samples were collected in Nellore (n= 6; body weight (BW) = 373 ± 37·3 kg) and Angus (n= 6; BW = 383 ± 23·9 kg) cattle at slaughter for analysis of gene and protein expression, and Calpastatin enzyme activity. Additionally, the myofibrillar fragmentation index was used to quantify the extension of proteolysis in longissimus muscle samples. A greater myofibrillar fragmentation was observed in skeletal muscle of Angus compared with Nellore cattle. Conversely, no differences were found between breeds for mRNA expression of Calpain 1 (CAPN1) and Calpastatin (CAST). Similarly, no differences were observed for the abundance of Calpain and Calpastatin proteins between skeletal muscles of Nellore and Angus cattle. Despite the lack of differences in mRNA and protein abundance, a greater activity of Calpastatin was observed in skeletal muscle of Nellore compared with Angus cattle. These data indicate that the greater proteolysis in skeletal muscle of Angus compared with Nellore cattle is mainly driven by a greater Calpastatin activity rather than Calpain or Calpastatin mRNA and protein expression.