Betacellulin (BTC) is a member of the epidermal growth factor family. It has two biological activities: mitogenic activity in fibroblasts and vascular smooth muscle cells, and differentiation activity for the differentiation of pancreatic acinar AR42J cells into insulin-secreting cells. The previous finding that recombinant BTC promotes the neogenesis of β-cells in a mouse model supports the possibility that BTC is a therapeutic protein. However, the mitogenic activity of BTC may not be needed for differentiation into β-cells and may cause a side effect in clinical use. We prepared several derivatives of BTC to segregate the two activities, to decrease the mitogenic activity, and to maintain the differentiation activity. We succeeded in obtaining BTC derivatives segregated by the two biological activities by preparing truncated-type derivatives. A derivative of BTC, BTC24–76, with a truncated N-terminal 23 amino acids and C-terminal 4 amino acids, was 2.5-fold more active in differentiation and had one-tenth of the mitogenic activity. The derivatives described in the present study should be helpful in future applications as therapeutic proteins and in basic research for discovery of a BTC-specific receptor.