Objective: This study aimed to assess the effect of fluoride varnish on glass ionomer microhardness changes after endogenous acid erosion challenge. Methodology: In this study, 40 conventional glass ionomer (CGI; Fuji IX) and 40 resin-modified glass ionomer (RMGI; Fuji IILC) discs were fabricated and divided into 4 subgroups (n ¼ 10) for immersion in synthetic gastric acid or saliva for 27 h with/without fluoride varnish application. The surface microhardness was measured at baseline and after immersion, and the change in microhardness was calculated. Data were analyzed using analysis of variance and T-test. Results: A reduction in microhardness was noted in all subgroups following immersion. The lowest change in microhardness of both CGI and RMGI occurred in artificial saliva. In CGI groups, the highest reduction in microhardness occurred in synthetic gastric acid with fluoride varnish application, and the reduction was significantly different from that of the CGI group with fluoride varnish application (p value ¼ .01). In RMGI groups, the highest reduction in microhardness was noted in synthetic gastric acid without fluoride varnish application, and the reduction was significantly different from that of the other groups (p value < .05). Conclusions: Exposure to synthetic gastric acid caused a significant reduction in microhardness of RMGI. Varnish application significantly decreased the acid susceptibility of RMGI, but not that of CGI.