The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR’ complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.