Usually, only focused femtosecond (fs) lasers at near-infrared (NIR) range can induce photo-damage to transparent cells, making it difficult to treat large amount of cells by such optical methods for photostimulation. In this study, we clarify the mechanism of photodamage to cells that are co-cultured with gold nanorods (GNRs) by fs laser. The pulse duration and repetition rate of the fs laser play a key role in cell damage suggesting that the heat accumulation con-tributes to the major part for the cell damage rather than the high peak power which mainly determines the efficiency of multiphoton excitation. We further show that cellular Ca2t can also be released in this scheme, but the process is more sensitive to peak power. Our results can provide a large-scale GNR-mediated photostimulation for cell signaling modulation.