Transplantation of cardiomyocytes derived from stem cells is a promising option for cardiac repair. However, how to obtain efficient cardiomyocytes from stem cells is still a great challenge. Understanding of the mechanism that regulates the cardiac differentiation of stem cells is necessary for the effective induction of cardiomyocytes. A clonal derivative named P19CL6 cells can easily differentiate into cardiomyocytes with 1% dimethyl sulfoxide (DMSO) treatment, which offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomics were performed to identify proteins associated with cardiomyocytes differentiation of P19CL6 cells induced by DMSO. Out of 543 non-redundant proteins identified, 207 proteins showed significant changes during differentiation with ≥1.2-fold or ≤0.83-fold changes cut-offs. Nine proteins were confirmed by the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis respectively. Notably, broad consistency was well showed between mRNA and protein expression for down-regulation of nucleosome assembly protein 1-like 1 (Nap1l1). Further study revealed that knockdown of Nap1l1 by stable transfection of shRNA vector significantly accelerated DMSO-induced cardiomyocytes differentiation of P19CL6 cells characterized by increases in expression of cardiac specific transcription factors, genes, and proteins (GATA4, MEF-2C, ANP, BNP, cTNT, and β-MHC). Therefore, Nap1l1 is a novel protein that regulates cardiomyocytes differentiation of P19CL6 cells induced by DMSO. J. Cell. Biochem. 113: 3788–3796, 2012. © 2012 Wiley Periodicals, Inc.