The Hedgehog (Hh) signaling pathway regulates the differentiation of many kinds of cells and plays a critical role in many embryonic and postnatal developmental processes. Gli1 and Gli2 are two transcription factors of the Hh signaling pathway. In this study, we used quantitative real-time polymerase chain reaction to detect the relative expression of Gli1 and Gli2 in 13 tissues from three two-year-old purebred Qinchuan cattle, as well as in different cell populations derived from muscle and different stages of myogenic differentiation of myoblasts. The expression levels of Gli1 and Gli2 in muscle were the lowest of the 13 tissues (P < 0.05), and they declined predominantly from preplate (pp)1 to pp6 cells. However, the expression of Gli2 was elevated during myogenic differentiation until the 6th day. We speculated that Hh signaling was negatively activated in myocytes and quiescent myoblasts. The increased expression of Gli1 and Gli2 in the early days of myogenic differentiation suggested that Hh signaling would be activated when the quiescent bovine myoblast was stimulated to initiate myogenic differentiation.