The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which domains are necessary. Prior studies suggested that the Pro-Ala rich region of human cMyBP-C activated force in permeabilized human cardiomyocytes, whereas the C1 and M-domains of mouse cMyBP-C activated force in permeabilized rat cardiac trabeculae. Because the amino acid sequence of the P/A region differs between human and mouse cMyBP-C isoforms (46% identity), we investigated whether species-specific differences in the P/A region could account for differences in activating effects. Using chimeric fusion proteins containing combinations of human and mouse C0, Pro-Ala, and C1 domains, we demonstrate here that the human P/A and C1 domains activate actomyosin interactions, whereas the same regions of mouse cMyBP-C are less effective. These results suggest that species-specific differences between homologous cMyBP-C isoforms confer differential effects that could fine-tune cMyBP-C function in hearts of different species.