An ethanol extract of the root of Cannabis sativa L., after partitioning and repeated chromatography, afforded a new spermidine (pyrido[2,1-d][1,5,9]triazacyclotridecine) alkaloid, cannabisativine (I), with the chemical name 13-(1,2-dihydroxyheptanyl)-1,5,6,7,8,9,10,11,13,16,16a-dodecahydropyrido[2,1-d][1,5,9]triazacyclotridecin-2(3H)-one. Crystallization of I from acetone gave white plates with mp 167–168°C and [α]₂₅ᴰ +55.1° (c 0.53, CHCl₃). Single-crystal X-ray diffraction data were collected on an automated Siemens diffractometer with Ni-filtered CuKα radiation, revealing space group P2₁2₁2₁ (cell constants a=23.70, b=13.43, c=7.26 Å), four molecules per unit cell, and a density (1.097 g/cc) consistent with calculated values (1.095 g/cc). The structure was solved via the direct method program Phase DT2 using 1682 independent reflections (1486 observed), with refinement yielding a final R factor of 8.8% after including 30 hydrogen atoms. Spectroscopic data supported the structure: 100 Hz NMR showed 2 vinyl protons (singlet at δ5.96) and an amide proton (broad singlet at δ9.6); IR indicated amide absorption at 1628 cm⁻¹; high-resolution mass spectrometry confirmed fragmentation pathways via strong metastable peaks. To our knowledge, this is the first report of a non-quaternary alkaloid from C. sativa roots—while similar pyrido[1,2-d][1,5,9]triazacyclotridecine alkaloids (palustrine, palustridine) were previously isolated from Equisetum (Equisetaceae), this is their first occurrence in a higher plant. X-ray analysis revealed hydrogen bonds (N3-N9, C18-OH-N12) and relative configuration: C17 and C1 are trans, C11 (off N12) is cis to C1, and alcohols at C17/C18 are erythro. Thin-layer chromatography detected cannabisativine in C. sativa leaves.