Light plays an important role in the life cycle of the myxomycete Physarum polycephalum, though the nature of its blue-light receptor remains unknown. HPLC analysis revealed that light exposure stimulates the formation of two metabolites in P. polycephalum plasmodia. Here we report the isolation and structure elucidation of these metabolites, named polycephalin C (1) and B (2). Isolation was performed under strict light exclusion and low temperature (4 °C) to preserve their stability. Structural characterization utilized HPLC, UV-Vis, IR, NMR (HMBC, HMQC), circular dichroism (CD), high-resolution mass spectrometry, and synthetic model compounds. Polycephalin C (1) has the molecular formula C32H36N2O8, consisting of two N-methyltetramic acid units linked via a cyclohexene ring with triene chains; CD analysis of its hydrogenated product confirmed S configuration at both C-5 positions. Synthetic isomeric diols (3, 4) verified the cyclohexene ring's relative stereochemistry. Polycephalin B (2) is the N-demethylated derivative of 1. Additionally, physarorubinic acid A (5) and a new polyenetetramic acid (physarorubinic acid B, 6) were identified. Polycephalin C (1) is highly light-sensitive and transforms under illumination, with further studies on its biosynthesis and photoproduct to be reported elsewhere.