Menthol is a widely used flavor additive in Electronic cigarette (E-cig) products and has been reported to act as a negative allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs). Lately, nicotine analogs such as 6-methylnicotine (6-MN) have emerged in E-cig products marketed as “nicotine-free” alternatives. Recent studies suggest these compounds may retain or enhance pharmacological activity at nAChRs, although their agonistic activity remains poorly characterized. In this study, we investigated the differential effects of menthol on nicotine- and 6-MN-induced activation of α7 nAChRs in in vitro and in vivo. We also examined synergistic interactions between nicotine and 6-MN. For in vitro assay, Ca²⁺ assay was performed using transfected HEK293T cells. Potency and efficacy were determined as EC₅₀ and E_max values relative to acetylcholine. For in vivo, a nose-poke self-administration model was conducted to allow mice to voluntarily inhale vaporized compounds. In vitro and in vivo results showed that menthol significantly reduced nicotine-induced α7 nAChRs activation, whereas menthol had no significant effect on 6-MN-induced responses. Furthermore, co-treatment of nicotine and 6-MN produced a synergistic interaction in α7 nAChRs activation in vitro. These findings indicate that nicotine analogs may still exert substantial pharmacological effects on α7 nAChRs, raising concerns regarding their safety and regulatory oversight.