Ailanthus altissima (Mill.) Swingle (Simaroubaceae) is a large tree ("Tree of Heaven") distributed from Southeast Asia to China and Taiwan, and also grown in European and North American cities. It is used as an antibacterial, anthelmintic, amoebicide, and insecticide, with biological activities mainly dependent on quassinoids and indole alkaloids (simple β-carbolines or canthin-6-ones). Its cell cultures are of particular interest for producing high yields of canthin-6-one alkaloids, which are normally minor constituents. The purpose of this study was to detailedly investigate the secondary metabolites produced by callus cultures of A. altissima. Freeze-dried callus cultures of A. altissima were sequentially extracted with petroleum ether and chloroform. From the crude chloroform extract, 6-β-OH-stigmasta-4-en-3-one (1) and 6-β-OH-stigmasta-4,22-dien-3-one (2) were isolated via silica gel column chromatography (CC) and reverse-phase HPLC, and no previous reports on their occurrence in cell cultures were found. Several indole alkaloids were isolated from the chloroform extract, including known constituents (canthin-6-one, 1-methoxycanthin-6-one, 1-hydroxycanthin-6-one, canthin-6-one-3-N-oxide, and 4-methoxy-β-carboline-1-carboxylic acid methyl ester, which had been reported in callus and cell suspension cultures of A. altissima before) and a new compound, 4,8-dimethoxy-β-carboline-1-carboxylic acid methyl ester (3). The 1H NMR data of 3 refer to [20], and its 13C NMR data are presented here for the first time. This study confirmed the production of a range of β-carboline and canthin-6-one alkaloids in callus cultures of A. altissima. The production of sterones in plant cell cultures has not been well studied, and plant tissue cultures of A. altissima might be important for further investigations on their biosynthesis and biological functions.