The purpose of this study was to evaluate the in vitro dentin bond strengths of two dentin bonding systems in Class I cavities following fatigue load cycling (FLC) with thermal cycling (TC). Class I cavities were prepared in 12 human molars and restored with either Clearfil SE Bond (SE) and Clearfil AP-X resin composite (AP-X) or Single Bond (SB)/AP-X according to the manufacturers’ instructions. After water storage for 1 week, untreated controls and test samples were subjected to 50,000 FLC with TC from 5°C to 55°C for 625 cycles and stored in water for 1 week. Microtensile bond strength (MTBS) tests were measured on the floor of the cavities at a crosshead speed of 1 mm/min. Data were statistically analyzed using one- and two-way ANOVA and Fisher’s PLSD test at a 5% level of significance. The MPa results were: SE 30.5 ± 12.8 (control) and 23.2 ± 10.8 (50,000 FLC); SB 5.1 ± 9.2 (control) and 4.7 ± 5.7 (50,000 FLC). Bond strengths were not statistically significantly affected by FLC (p > 0.05); however, they were influenced by the bonding system (p < 0.05). The MTBS of SE was significantly higher than that of SB (p < 0.05) under FLC and TC test conditions. Over half of the SB specimens debonded during sample preparation, showing blister-like structures, suggesting that an over-wet phenomenon occurred on the cavity floor. SE produced excellent dentin bonds that were much stronger than those produced by SB, which was shown to be a technique-sensitive bonding system.