Nitro-fatty acids (NO2-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO2-FAs are considered novel signaling mediators of cell function based on a proven anti-inflammatory response. Nevertheless, the interaction of NO with fatty acids in plant systems has been scarcely studied. Here, we examine the endogenous occurrence of nitro-linolenic acid (NO2-Ln) in Arabidopsis and the modulation of NO2-Ln levels throughout this plant's development by mass spectrometry. The observed levels of this NO2-FA at picomolar concentrations suggested its role as signaling effectors of cell function. In fact, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO2-Ln was involved in plant defense response against different abiotic-stress conditions, mainly by inducing heat shock proteins (HSPs) and supporting a conserved mechanism of action both in animal and plant defense processes. Moreover, bioinformatics analysis revealed that NO2-Ln was also involved in the response to oxidative stress conditions, mainly depicted by H2O2, ROS and oxygen-containing compound responses, with a high induction of ascorbate peroxidase (APX) expression. Closely related to these results, NO2-Ln levels significantly rose under several abiotic-stress conditions such as wounding or exposure to salinity, cadmium, and low temperature thus validating the outcomes found by RNA-seq technology. Jointly, these are the first results showing the endogenous presence of nitro-linolenic acid in Arabidopsis and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.