So far, nitric oxide (NO) donors have been applied to various aspects of antitumor therapy. To selectively sensitize tumor cells and avoid unwanted side effects, we recently synthesized a beta-galactosidase-activatable NO-releasing compound, beta-galactosyl-pyrrolidinyl diazeniumdiolate (beta-Gal-NONOate). In this study, we first verified its superiority over its parent diazeniumdiolate (NONOate) in terms of targeted intracellular NO-releasing and antitumor activity with 9L/LacZ cells (rat glioma cell line 9L with transformed LacZ gene) in vitro. beta-Gal-NONOate only released NO when hydrolyzed by induced beta-galactosidase in 9L/LacZ cells, which led to its more powerful cytotoxicity than that of NONOate. The results showed that beta-Gal-NONOate produced higher NO levels than NONOate in 9L/LacZ cells at equal concentration, and hence induced optimal NO levels for antitumor activity. However, in 9L cells, beta-Gal-NONOate showed less toxicity than NONOate. Therefore, it is demonstrated that beta-Gal-NONOate is a site-specific prodrug for targeting NO intracellularly as a beta-galactosidase-sensitive NO donor, and it is also expected to be a promising probe in numerous experimental settings and a potential therapeutic drug for antitumor treatment.