Some N-acyl dopamines have been reported to show anti-inflammatory and immunomodulatory activities. In this respect, we examined the immunosuppressive effect of N-acyl dopamines on splenocytes of BALB/c mice. First, when the effect of each endocannabinoid or its derivative on Con A-induced proliferation of splenocytes, N-arachidonoyl dopamine (NADA), N-oleoyl dopamine (OLDA) and N-palmitoyl dopamine (PALDA) potently suppressed Con A-mediated splenocyte proliferation with IC50 value of 1.84, 2.45, and 8.54 µM, respectively, indicating that NADA and OLDA were more immunosuppressive than PALDA. In related study, the immunosuppressive effect of NADA or PALDA was antagonized partially by CB1 antagonist, SR141716, and TRPV1 antagonist, 5′-iodoresiniferatoxin (5′-IRTX), but not CB2 antagonist, AM630. Meanwhile, the effect of OLDA was suppressed partially by 5′-IRTX, but not other antagonists. In further study to support the immunosuppressive activity of N-acyl dopamines, the effect of N-acyl dopamaines on the release of cytokines was investigated. Noteworthy, NADA (IC50, 0.53 µM), OLDA (IC50, 0.78 µM) and PALDA (IC50, 2.45 µM) inhibited the secretion of interferon-γ from Con A-stimulated splenocytes concentration-dependently. A similar suppression of the release of tumor necrosis factor alpha-α and interleukin-2 was also expressed by N-acyl dopamines. Additionally, the suppressive effect of NADA or PALDA on cytokine release was antagonized partly by SR141716 or 5′-IRTX, and that of OLDA was antagonized partly by 5′-IRTX. Separately, NADA and OLDA enhanced the level of 12(S)-hydroxyeicosatetraenoic acid an endogenous TRPV1 agonist. These findings suggest that N-acyl dopamines may express immuno-modulatory action through pathways involving CB1 and TRPV1 receptors in splenocytes.