In the present study, the enhancing effect of 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP) on the percutaneous absorption of indomethacin (IM) was evaluated by the in vitro penetration experiments using the rat abdominal skin as a barrier. Partition experiment, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum and transepidermal water loss (TEWL), was employed to investigate the possible mechanisms of the action of M-HEP. Furthermore, the reversible effect of M-HEP on excised rat skin was also evaluated through in vitro permeation as a preliminary indicator of safety. The result of in vitro permeation experiment indicated that, 10% (w/w) M-HEP in combination with isopropyl palmitate (IPP) significantly increased (p < 0.05), the cumulative amount of IM in comparison with the control group (IPP only). The partition coefficient of IM between the stratum corneum (SC) and enhancer solution was also greater than that between the SC and IPP. A blue shift in the ATR-FTIR spectra of SC after treatment with M-HEP solution was observed at the CH(2) band, which indicating that M-HEP disrupted the intercellular lipid structure of the SC. In addition, both M-HEP/IPP and L-menthol (MT)/IPP improved the TEWL value of rat abdominal skin. After removal of M-HEP, the skin barrier function would be restored in 8 h. In conclusion, M-HEP could reversibly enhance the percutaneous absorption of IM by increasing the partitioning of IM into the SC from enhancer solution and disturbing the organized structure of SC lipids and the reversibility of M-HEP was better than MT.