The 5-HT7 receptor (5-HT7R) is a promising therapeutic target for the treatment of depression and neuropathic pain. The 5-HT7R antagonist SB-269970 exhibited antidepressant-like activity, whereas systemic administration of the 5-HT7R agonist AS-19 significantly inhibited mechanical hypersensitivity and thermal hyperalgesia. In our efforts to discover selective 5-HT7R antagonists or agonists, aryl biphenyl-3-ylmethylpiperazines were designed, synthesized, and biologically evaluated against the 5-HT7R. Among the synthesized compounds, 1-([2′-methoxy-(1,1′-biphenyl)-3-yl]methyl)-4-(2-methoxyphenyl)piperazine (28) was the best binder to the 5-HT7R (pKi=7.83), and its antagonistic property was confirmed by functional assays. The selectivity profile of compound 28 was also recorded for the 5-HT7R over other serotonin receptor subtypes, such as 5-HT1R, 5-HT2R, 5-HT3R, and 5-HT6R. In a molecular modeling study, the 2-methoxyphenyl moiety attached to the piperazine ring of compound 28 was proposed to be essential for the antagonistic function.