A major challenge in stem cell therapy for cardiac repair is how to obtain normally functioning stem cell-derived cardiomyocytes. We aim to address the effects of C-reactive protein (CRP) on the cardiac differentiation of embryonic stem (ES) cells. Immunostaining, Western blotting and electrophysiology were employed. A hundred fifty milligran/liters CRP significantly reduced the percentage of cardiomyocytes differentiated from mouse ES cells, while it may also promote sarcomere development compared to 30 mg/L CRP treatment. Further examination of the action potential (AP) in individual ES cell-derived cardiomyocytes showed that there exist three types of cardiomyocytes: artial-like (A-like), ventricular-like (V-like), and pacemaker-like (P-like). A hundred fifty milligran/liters CRP treatment decreased the P-like cardiomyocytes, whereas it increased the A-like. Such inhibitory effect and alteration were not significant at 30 mg/L CRP treatment. Moreover, 150 mg/L CRP significantly increased the APD90 (90% of duration of AP) and decreased the spontaneous firing rate of AP in P-like cells, while had little effect on other electrophysiological characteristics, including APA (AP amplitude) and MDP (maximum diastolic potential). This study revealed the effect of CRP on the cardiac differentiation of ES cells. It provides an in vitro pathological model and may be of importance to the future work of ES cell-based therapy in clinical applications and in vivo pathological studies.