Nitrogen (N) is the primary limiting factor for crop growth, development, and productivity. Transgenic technology is a straightforward strategy for improving N assimilation in crops. The present study assessed the effects of maize C4 phosphoenolpyruvate carboxylase (ZmPEPC) gene overexpression on N assimilation in three independent transgenic lines and wild-type (WT) wheat (Triticum aestivum L.). The transgenic wheat lines depicted ZmPEPC overexpression and higher PEPC enzyme activity relative to that in the WT. The leaves of the transgenic wheat lines subjected to low N treatment showed an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expression, content, and carboxylase activity. The transgenic wheat lines also depicted an upregulation of genes associated with the anaplerotic pathway for the TCA cycle, suggesting that more carbon (C) skeleton material is being allocated for N assimilation under low N conditions. Furthermore, ZmPEPC expression in transgenic wheat lines induced the upregulated of genes associated primary N metabolism, including TaNR, TaGS2, TaGOGAT, TaAspAT, and TaASN1. The average total free amino acid content in the transgenic wheat lines was 48.18% higher than that in the WT, and asparagine (Asn), glutamine (Gln), aspartic acid (Asp), and serine (Ser) were also markedly enhanced. In addition, elementary analysis showed that N and C content, and the biomass of the transgenic wheat lines increased with low N treatment. Yield trait analysis indicated that ZmPEPC overexpression improved grain yield by increasing 1000-grain weight. In conclusion, ZmPEPC overexpression in wheat could modulate C metabolism, significantly improve N assimilation, enhances growth, and improves yield under low N conditions.