Vitamin K1 (phylloquinone) is a substituted membrane-anchored naphthoquinone that functions as an essential electron carrier in photosystem I in photosynthetic organisms. While plants can synthesize phylloquinone de novo, humans rely on vitamin K1 uptake from green leafy vegetables as a precursor for the synthesis of its structural derivative, menaquinone-4 (vitamin K2). In vertebrates, menaquinone-4 serves as an enzymatic co-factor that is required for posttranslational protein modification, i.e. the γ-carboxylation of glutamate residues in specific proteins involved in blood coagulation, bone metabolism and vascular biology. Comprehensive knowledge of the subcellular compartmentalization of vitamin K biosynthesis in plants, pathway regulation and its integration in cellular metabolic networks is important to design functional food with elevated vitamin levels and health benefits to human consumers. It had long been assumed that plants obtained all enzymes for phylloquinone biosynthesis from the ancient cyanobacterial endosymbiont and that, upon gene transfer to the nucleus, all biosynthetic enzymes were re-directed to the plastid. This view, however, has been recently challenged by the exclusive localization of the 6th pathway enzyme (MenB/NS) to peroxisomes in Arabidopsis. Soon afterwards, not only the preceding enzyme, acyl-activating enzyme 14 (MenE/AAE14), but also the succeeding thioesterase (DHNAT) were also shown to be peroxisomal. Phylogenetic analysis revealed a heterogeneous evolutionary origin of the peroxisomal enzymes. Phylloquinone biosynthesis reveals several branching points leading to the synthesis of important defence signalling molecules, such as salicylic acid and benzoic acid derivatives. Recent research data demonstrate that, of the two phenylalanine-dependent pathways for benzoic and salicylic acid biosynthesis, the CoA-dependent β-oxidative pathway, which is peroxisomal, is the major route. Hence, peroxisomes emerge as an important cell compartment for the interconnected networks of phylloquinone, benzoic and salicylic acid biosynthesis. Numerous mechanisms to regulate intermediate flux and the fine-tuned inducible production of secondary metabolites, including signalling molecules, await their characterization at the molecular level.