Potassium (K+) is an essential macronutrient for plant growth, development, and fruit quality and yield. K+ uptake and transport is facilitated by KT/HAK/KUP transporters. However, studies to establish molecular mechanisms are rare in fruits, especially in peaches. In this study, we isolated 16 putative KT/HAK/KUP transporter genes in peach, and analyzed K+ homeostasis status in relation to KUP (K+ uptake) gene expression during whole fruit development life. The ‘Xiahui6’ peach development was divided into four distinct stages, S1–S4, and fruits were harvested on 110 days after full bloom (DAFB). QRTPCR results showed that PpeKUP genes unevenly existed in various fruit parts and are differentially expressed during fruit development, ripening, and postharvest storage. The most highest-expressed gene was PpeKUP1 in mesocarp and PpeKUP2 in skin, especially during early stages, while PpeKUP3 was steadily expressed even until postharvest shelf-life. After harvest, the flesh firmness was nonsignificantly changed under cold treatment (4 C), to avoid ripening. Notably, five PpeKUP genes were responsive to cold treatment as their expression were mainly induced in skin, except for PpeKUP3 that was decreased in both mesocarp and skin. Moreover, functional determination showed that PpeKUP1 and PpeKUP2 are important K+ transporters that mediate K+ uptake and accumulation, especially during fruit formation and fast growth stages. This study reveals a close relationship among peach growth, firmness maintenance, and K+ homeostasis, and directly provides potential candidate genes for further molecular studies.