High gestational weight gain (GWG) is a cardiovascular risk factor and may disturb neonatal endothelial function. Long non‐coding RNAs (lncRNAs) regulate gene expression epigenetically and can modulate endothelial function. Endothelial colony forming cells (ECFCs), circulating endothelial precursors, are a recruitable source of endothelial cells and sustain endothelial function, vascular growth and repair. We here investigated whether higher GWG affects neonatal ECFC function and elucidated the role of lncRNAs herein. Wound healing of umbilical cord blood‐derived ECFCs after pregnancies with GWG <13 kg versus >13 kg was determined in a scratch assay and based on monolayer impedance after electric wounding (electric cell‐substrate impedance sensing, ECIS). LncRNA expression was analysed by RNA sequencing. The function of killer cell lectin‐like receptor K1 antisense RNA (KLRK1‐AS1) was investigated after siRNA‐based knockdown. Closure of the scratch was delayed by 25% (P = 0.041) in the higher GWG group and correlated inversely with GWG (R = −0.538, P = 0.012) in all subjects (n = 22). Similarly, recovery of the monolayer barrier after electric wounding was delayed (−11% after 20 h; P = 0.014; n = 15). Several lncRNAs correlated with maternal GWG, the most significant one being KLRK1‐AS1 (log₂ fold change = −0.135, P < 0.001, n = 35). KLRK1‐AS1 knockdown (n = 4) reduced barrier recovery after electric wounding by 21% (P = 0.029) and KLRK1‐AS1 expression correlated with the time required for wound healing for both scratch (R = 0.447, P = 0.033) and impedance‐based assay (R = 0.629, P = 0.014). Higher GWG reduces wound healing of neonatal ECFCs, and lower levels of the lncRNA KLRK1‐AS1 may underlie this.