Aims: The effect of mechanical loading on the microtensile bond strength (μTBS) of universal adhesives to dentin was evaluated in this study. Methods and Materials: Human molar teeth had the occlusal dentin surfaces exposed and were allocated into ten groups (five experimental groups and five control groups) that used the following universal adhesive systems in self-etch mode: All-Bond Universal (ABU), Single Bond Universal (SBU), Gluma Bond Universal (GBU), Tetric N-Bond Universal (TBU), and Clearfil Universal Bond (CUB). Following the bonding procedures and build-ups, the specimens were either stored in water at 37°C for 24 h or were mechanically loaded (50 N for 60,000 cycles) prior to the μTBS test. Data were analyzed using the one-way analysis of variance (ANOVA) and Tukey's posthoc test (P = 0.05). Results: Both the adhesive type and mechanical loading had significant effects on the μTBS (P < 0.05). The μTBS values of SBU and ABU were significantly higher than the values of the other adhesives (P < 0.05). However, the μTBS values of ABU decreased significantly after mechanical loading (P < 0.05). Conclusions: With the exception of ABU, mechanical loading had no deleterious effects on the μTBS of the universal adhesive systems examined in this study.