Zein, a storage protein rich in hydrophobic amino acids found in maize endosperm, offers significant potential for applications in food packaging. Previous studies on zein films primarily focused on the dissolution of zein in organic solvents. In this study, zein was dissolved in an alkaline solution instead of organic solvents. The effects of fatty acids with varying carbon chain lengths, namely decanoic acid, lauric acid, and palmitic acid, on the structure and properties of zein films were investigated. Alkali-solubilized zein exhibited a various degree of deamidation related to fatty acids after film formation in which decanoic acid significantly reduced the deamidation of zein, while palmitic acid had the opposite effect. Various characterizations confirmed that fatty acids were evenly distributed within the film matrix, disrupting the intermolecular interactions of zein and preventing zein molecules from aggregation; this eventually led to a tightly structured film. The addition of fatty acids, specifically decanoic acid and lauric acid, significantly enhanced the performance of the zein films. After loading decanoic acid or lauric acid, the almost opaque zein films had a visible light transmittance of over 50% while maintaining an excellent UV light blocking property. The tensile strength and elongation at break of the films were improved, and their water vapor permeability was reduced by an order of magnitude. This study provides insights into zein plasticization and paves the way for developing organic solvent-free food packaging.