Thymoquinone (TQ) is the bioactive constituent of the volatile oil of Nigella sativa. The chemotherapeutic and chemopreventive effects of Nigella sativa seeds are mainly attributed to TQ. TQ has been shown to exert anti-inflammatory, antioxidant and antineoplastic effects both in vitro and in vivo. The anti-angiogenic effects of TQ were evaluated using in vivo zebrafish angiogenesis model. To investigate the anti-angiogenic activity of TQ, we tested its inhibitory effect on blood vessel formation followed by elucidation of its molecular target in zebrafish embryos. Our results showed that TQ inhibited the growth of intersegmental vessel (ISV) of zebrafish embryos in a dose-dependent manner, as observed by red blood cells (RBC) staining assay. TQ was also found to down-regulate the expression of VEGF-A mRNA. This study reveals the potential anti-angiogenic effect of TQ, thus proving its role as a therapeutic agent against diseases like cancer.