The force-generating capacity of cardiomyocytes rapidly changes during gestation and early postnatal life coinciding with a transition in cardiomyocyte nucleation in both mice and rats. Changes in nucleation, in turn, appear to coincide with important changes in the excitation-contraction coupling architecture. However, it is not clear whether similar changes are observed in other mammals in which this transition occurs prenatally, such as sheep. Using small (70–300 μM diameter) chemically skinned cardiomyocyte bundles from the right ventricular papillary muscle of sheep fetuses at 126–132 and 137–140 days (d) gestational age (GA), we aimed to examine whether changes in cardiomyocyte nucleation during late gestation coincided with developmental changes in excitation-contraction coupling parameters (e.g., Ca2+ uptake, Ca2+ release, and force development). All experiments were conducted at room temperature (23 ± 1°C). We found that the proportion of mononucleate cardiomyocytes decreased significantly with GA (126–132d, 45.7 ± 4.7%, n = 7; 137–140d, 32.8 ± 1.6%, n = 6; P < 0.05). When we then examined force development between the two groups, there was no significant difference in either the maximal Ca2+-activated force (6.73 ± 1.54 mN/mm2, n = 14 vs. 6.55 ± 1.25 mN/mm2, n = 7, respectively) or the Ca2+ sensitivity of the contractile apparatus (pCa at 50% maximum Ca2+-activated force: 126–132d, 6.17 ± 0.06, n = 14; 137–140d, 6.24 ± 0.08, n = 7). However, sarcoplasmic reticulum (SR) Ca2+ uptake rates (but not Ca2+ release) increased with GA ( P < 0.05). These data reveal that during late gestation in sheep when there is a major transition in cardiomyocyte nucleation, SR Ca2+ uptake rates increase, which would influence total SR Ca2+ content and force production.