For the biosynthesis of Pip in Huperzia serrata, the mechanistic studies were evaluated. Through a series of biochemical analyses, Pip is biosynthesized through a two-step cascade reaction. Three intermediates possibly exist simultaneously as an equilibrium matter in the first-step reaction catalyzed by HsAld1, while HsSard4 performs as a ketimine reductase and chemoselectively and stereoselectively takes 1,2-dehydropipecolic acid as the preferred substrate in vitro.