Bone marrow-derived mesenchymal stem cells (BM-MSCs) contribute to myocardial repair after myocardial infarction (MI) by secreting a panel of growth factors and cytokines. This study was to investigate the potential mechanisms of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) in regulation of the profiles of BM-MSCs secretion and compare the therapeutic efficacy of NUCKS-/-- and wide type-BM-MSCs (WT-BM-MSCs) on MI. The secretion profiles between NUCKS-/-- and WT-BM-MSCs under hypoxia (1%O2) were analyzed. Gene function analysis showed that compared with WT-BM-MSCs-conditioned medium (CdM), some genes over-presented in NUCKS-/--BM-MSCs-CdM were closely associated with inflammatory response, regulation of cell proliferation, death, migration and secretion. Notably, VEGFa in NUCKS-/--BM-MSCs-CdM was higher than that of WT-BM-MSCs-CdM. WT-BM-MSCs and NUCKS-/--BM-MSCs were transplanted into the peri-infarct region in mice of MI. At 4 weeks after cell transplantation, NUCKS-/-- or WT-BM-MSCs group significantly improved heart function and vessels density and reduced infarction size and apoptosis of cardiomyocytes. Furthermore, NUCKS-/--BM-MSCs provided better cardioprotective effects than WT-BM-MSCs against MI. Our study demonstrates that depletion of NUCKS enhances the therapeutic efficacy of BM-MSCs for MI via regulating the secretion.