The erbium-doped yttrium aluminum garnet (Er: YAG) laser has been successfully applied in caries removal; however, little is known about proper parameters of Er: YAG laser on different conditions of caries removal, especially the influence of Er: YAG irradiation on human dental pulp cells (hDPCs). Here, we tested the effects of Er: YAG laser at different output energy levels (100, 200, 300, 400, and 500 mJ) on biobehaviors of hDPCs. To simulate clinical deep caries conditions, hDPCs were cultured on the pulpal side of 500-μm-thick dentin disks in an in vitro pulp chamber model. Temperature change, structural change, and ablation depth of dentin disk were also recorded. The findings suggested that the biological behaviors of hDPCs are strongly correlated with the energy output of the Er: YAG laser. Er: YAG laser irradiation at 100 mJ may be proper and safe for deep caries removal since it would not cause any adverse effect on hDPCs biobehaviors.