This study analyzed the effect of gender on the abundances of 20 protein biomarkers of tenderness and/or intramuscular fat content in five muscles: Longissimus thoracis, previously identified as biomarkers of tenderness and/or intramuscular Semimembranosus, Rectus abdominis, Triceps brachii and Semitendinosus, from cows and steers of the Protected Designation Origin Maine Anjou. The protein abundances were quantified using Reverse Phase Protein Array with specific validated antibodies. Among the 20 studied proteins, the abundance of 8 biomarkers involved in energetic metabolism, contraction and cellular stress, was different according to gender. The gender effect was different depending on the muscle type with greater abundances in Semitendinosus, Rectus abdominis and Longissimus thoracis muscles. On the basis of animal characteristics and rearing factors, three rearing practices classes were identified for cows. Among the factors, fattening duration modified the abundance of 12 proteins mainly in Triceps brachii muscle. A positive correlation between the abundance of the small HSP20 and slaughter age was observed in the 5 muscles. Two proteins, Four and a half LIM domains 1 (FHL1) and Glycogen phosphorylase (PYGB) appeared to be muscle, gender and rearing practices independent. These results constitute valuable data to understand how to manage beef quality by controlling these different factors.