Emotional responses such as fear and anxiety are essential for decision-making and survival. The habenula (Hb) is a structure in the epithalamus highly conserved during vertebrate evolution. It mediates behavioral responses to stress, anxiety, and fear. Dysfunction of Hb is associated with depression, post-traumatic stress disorder, and schizophrenia in humans. Furthermore, the ablation of Hb has been shown to profoundly alter fear and anxiety responses in fish and mice. However, the molecular mechanisms underlying the emotional responses and functional regulation of Hb remain largely unknown. Here we report the critical function of a novel chemokine-like protein, samdori-2 (sam2), in anxiety and fear-related behaviors. We found that sam2 is predominantly expressed by neurons in the dorsal Hb of adult zebrafish. Targeted knockout (KO) of the sam2 gene by zinc finger nucleases in zebrafish did not affect viability, fertility, or general morphology. Furthermore, sam2 KO fish showed normal development of neural circuits including Hb to interpeduncular nucleus (IPN) pathways. However, sam2 KO fish exhibited robustly elevated anxiety-like behaviors in the novel tank and scototaxis tests, and showed increased social cohesion. In addition, nicotine-induced c-fos expression in the dorsal Hb-IPN pathway was significantly inhibited in the sam2 KO fish. Consistent with these findings, Sam2 was found to strongly attenuate inhibitory synaptic function in neurons. These results establish a crucial role of sam2 in regulating anxiety and social cohesion, and delineate a potential function of novel chemokine-like proteins in controlling neuronal circuit activity and behavior.