The purpose of this study was to evaluate the effect of 2% chlorhexidine (CHX) on an etch-and rinse adhesive to dentin. Caries-free molars were selected and processed to expose a flat dentin surface. The specimens were bonded with a composite resin by an etch-and-rinse adhesive, which was pretreated with 2% CHX for 0, 15, 30, 45, and 60 seconds. The microtensile bond strength (MTBS) was evaluated before and after thermocycling. No significant differences were observed between groups before thermocycling. The 60-second CHX-pretreated group showed a significantly greater MTBS than the control group after thermocycling. The 2% CHX pretreatment could improve the bonding strength of the etch-and-rinse adhesive and slow down the aging progress of the bonding interface.