Messenger RNA (mRNA) expression of calpain‐1 (µ‐calpain), ‐2 (m‐calpain), ‐3 (p94), small subunit (calpain‐4; 28 kDa), and three types of calpastatin (CSTN) isoform were investigated for 10 skeletal muscles of Holstein cattle by real‐time and/or semi‐quantitative reverse transcription polymerase chain reaction. Noticeably, effect of muscle type was observed on 28 kDa expression (P < 0.001) with a tendency of higher 28 kDa expression in myosin heavy chain (MyHC)‐2x‐rich muscles compared to MyHC‐slow‐rich muscles. The CSTN‐I and –III expression in Longissimus thoracis (LT) showed the lowest value among the muscles tested. Moreover, 28 kDa/CSTN‐I ratio was higher in the diaphragm (DP), psoas major (PM), and LT than those in the lingual muscles (TN), masseter (MS) and pectoralis (PP) (P < 0.05). Calpain‐1/CSTN I, calpain‐2/CSTN I in LT and PM were higher than that in TN (P < 0.05). Calpain‐3/CSTN‐I and ‐III in LT and/or PM showed higher values than that in TN (P < 0.05). These results indicated that the calpain and CSTN expressions are regulated by muscle type, suggesting especially by muscle fiber type. Calpains/CSTN‐I ratios, especially 28 kDa/CSTN‐I, may account for higher extent of post mortem proteolysis previously observed in LT and PM muscles.