A series of novel 9-substituted β-carboline derivatives was synthesized from harmine and L-tryptophan, respectively. Cytotoxic activities of these compounds in vitro were investigated. The results showed that most compounds of 9-substituted β-carboline derivatives had more remarkable cytotoxic activities in vitro than their corresponding parent compounds. Acute toxicities and antitumor effects of the selected β-carboline derivatives in mice were also examined. The results demonstrated that a short alkyl or benzyl substituent at position-9 increased the antitumor activities significantly and a ethoxycarbonyl or carboxyl substituent at position-3 reduced the acute toxicity and neurotoxicity of these β-carboline derivatives dramatically. Moreover the compounds both with an alkoxycarbonyl or carboxyl substituent at position-3 and a short alkyl or benzyl substituent at positon-9 exhibited more significant antitumor activities and lower acute toxicities and neurotoxicities than the other compounds. The compound 8c, having an n-butyl and a carboxyl substituent at position-9 and 3, respectively, was found to have the highest antitumor effect and the lowest acute toxicity and neurotoxicity. These data suggested that (1) appropriate substituents at both position-9 and 3 of β-carboline derivatives might play a crucial role in determining their enhanced antitumor activities and decreased acute toxicities and neurotoxic effects; (2) the β-carboline derivatives have the potential to be used as antitumor drug leads.