Quaternary alkaloids in Zanthoxylum nitidium were examined using ion-pair HPLC combined with ion-pair extraction. A new benzylisoquinoline alkaloid, (R)-(+)-isotembetarine (1), was isolated along with seven known alkaloids: chelerythrine, nitidine, (+)-magnoflorine, (+)-tembetarine, (+)-menisperine, (-)-cis-N-methylcanadine, and N,N,N-trimethyltryptamine. The structure of 1 was elucidated by HRLSIMS (molecular formula C20H26NO4+), UV, 1H/13C NMR (including 2D COSY, HMQC, HMBC, NOESY), and CD spectral data. Its absolute configuration at C-1 was determined as R by comparing CD spectra with (-)-oblongine. Known alkaloids were identified via authentic sample comparison or synthesis (for N,N,N-trimethyltryptamine). This study explores the water-soluble quaternary alkaloid constituents of Z. nitidium, a medicinal plant with antiinflammatory, analgesic, and bactericidal properties.