Pyruvate kinase (PK) is a key enzyme in glycolysis that regulates sugar metabolism and energy production, thereby influencing fruit quality. The ‘Feizixiao’ litchi, widely cultivated in Hainan Province, faces sugar reduction during fruit ripening. This study evaluated the effects of the foliar application of calcium and magnesium (Ca+Mg) during the fruit expansion stage to alleviate this problem. Ca+Mg foliar application significantly enhanced soluble sugar content, promoted peel coloration, and reduced respiration and PK activity. Genome-wide analysis identified 19 PK genes (LcPKs) exhibiting diverse exon-intron structures and conserved motifs. Phylogenetic analysis revealed both conserved and species-specific features, while subcellular localization predicted that most LcPK proteins are likely to be localized in the cytoplasm. Synteny analysis showed closer evolutionary relationships with species in the same genus than with Arabidopsis. Cis-regulatory element analysis implicated LcPKs in light response, hormone signaling, growth, and stress adaptation. Hormonal assays at 63 and 70 DAA after treatment revealed increased abscisic acid (ABA) and ethylene levels under Ca+Mg application. These hormonal changes correlated with the downregulation of LcPK3, LcPK4, LcPK5, LcPK8, and LcPK15, as confirmed by qRT-PCR, indicating negative regulation by ABA and ethylene. This regulatory mechanism likely contributes to overcoming sugar receding in litchi pulp. These findings offer insights into the regulation of sugar metabolism and strategies for enhancing fruit quality through the management of genes and nutrients.