Tiny predators, especially like phytoseiid mites, often experience a host of threats or stresses by fluctuating environmental factors. Heat acclimation as a superior adaptation strategy critically enhances abilities for organisms to handle with changing climate, but little is known about the molecular mechanism determining tolerant plastic responses in Phytoseiid mites. The relative expression of four identified HSP70 genes in two strains of Neoseiulus barkeri increased within a short time in temperature ramping treatment; meanwhile the expression of NbHSP70-1 and NbHSP70-2 in the conventional strain (CS) sharply decreased after 4 h displaying distinct contrast with the stable expression in the high-temperature adapted strain (HTAS). Western blot analysis showed that the protein level of NbHSP70-1 in CS was dramatically elevated at 0.5 h and decreased at 6 h at 42°C. Conversely, in HTAS, NbHSP70-1 was constantly induced and peaked at 6 h changed at 42°C. Furthermore, HSP70 suppression by RNAi knockdown had a greater influence on the survival of HTAS, causing a higher mortality under high temperature than CS. The recombinant certain exogenous NbHSP70-1 protein enhanced the viability of E. coli BL21 under lethal temperature of 50°C. These results suggested that HSP70 genes were a prominent contributor promoting the thermotolerance to heat stress and plastic change of HSP70 genes conferred the thermotolerance of HTAS through long-term heat acclimation. The divergent constitutive regulation of HSP70 to thermal is conducive to the flexible adaptability of predators in higher trophic level to trade off under extremely adversity stress.