Purpose: The purpose of this study was to evaluate the effect of the Er,Cr:YSGG laser on the enamel's acid resistance when irradiated alone, prior to or after acidulated phosphate fluoride (APF) treatment. Methods: Eighty-five enamel surfaces were prepared from 17 extracted human premolars and randomly assigned to 5 groups: (1) untreated (control); (2) irradiated with a Er,Cr:YSGG laser; (3) exposed to a 1.23% APF gel; (4) laser-irradiated followed by APF treatment; and (5) APF-treated followed by laser irradiation. The specimens were individually demineralized in 0.2 M acetate buffer solution for 10 days, and the acid resistance was evaluated by determining the calcium ion using atomic absorption spectrometry. Results: Significant differences were found between group 1 and groups 2 through 5 (P<0.001). There were no significant differences between groups 2 and 3 (P=1.000). Compared to groups 2 and 3, a lower calcium content was detected in the demineralization solution for groups 4 and 5. The difference between groups 4 and 5, however, was not statistically significant (P=0.289). Conclusions: Combining acidulated phosphate fluoride with the Er,Cr:YSGG laser decreased the enamel demineralization more than either fluoride treatment or laser treatment alone.