Fresh spinach (Spinacia oleracea L.) leaves contain high concentrations of beneficial phytochemicals. However, spinach does not store well and is highly perishable especially during handling and post-harvest storage. In this study, we investigated the effect of pre-harvest root-treatment of spinach with Ascophyllum nodosum extract (ANE) at different concentrations (0, 0.1, 1.0, or 5.0 g L−1) on post-harvest quality of fresh-cut spinach over a 35-day storage period. At the time of harvest there was no significant difference in dry weight, chlorophyll, ascorbate and lipid peroxidation between ANE-treated and non-treated plants. However, the loss in fresh weight and visual quality (color and turgor) of spinach leaves during storage was reduced by pre-harvest application of ANE. Lipid peroxidation was significantly reduced in ANE-treated leaves. The total chlorophyll content and ascorbate content in the control and treated leaves was however identical over the storage period and decreased at a similar rate. A negative correlation was observed between visual quality and lipid peroxidation. The results show that pre-harvest ANE application through root drench, especially at 1.0 g L−1, enhanced post-harvest storage quality of spinach leaves.