Saxitoxin, the most potent voltage-gated sodium channel blocker, is one of the paralytic shellfish toxins (PSTs) produced by cyanobacteria and dinoflagellates. Recently, putative biosynthetic genes of PSTs were reported in these microorganisms. We previously synthesized genetically predicted biosynthetic intermediates, Int-A′ and Int-C′2, and also Cyclic-C′ which was not predicted based on gene, and identified them all in the toxin-producing cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium tamarense (Axat-2). This study examined the incorporation of 15N-labeled intermediates into PSTs (C1 and C2) in A. circinalis (TA04). Conversions from Int-A′ to Int-C′2, from Int-C′2 to Cyclic-C′, and from Int-A′ and Int-C′2 to C1 and C2 were indicated using high resolution-LC/MS. However, Cyclic-C′ was not converted to C1 and C2 and was detected primarily in the extracellular medium. These results suggest that Int-A′ and Int-C′2 are genuine precursors of PSTs, but Int-C′2 converts partially to Cyclic-C′ which is a shunt product excreted to outside the cells. This paper provides the first direct demonstration of the biosynthetic route towards saxitoxin and a shunt pathway.