Research on deep-tissue photothermal therapy (PTT) in the near-infrared II (NIR-II, 1000-1350 nm) region has bloomed in recent years, owing to higher maximum permissible exposure and deeper tissue penetration over that in the near-infrared I (NIR-I, 650-950 nm) region. However, more details need to be uncovered to facilitate a fundamental understanding of NIR-II PTT. Herein, a tumor-targeted therapeutic nanosystem based on NIR-responsive molybdenum oxide (MoO 2) nanoaggregates was fabricated. The photothermal conversion capabilities of MoO 2 in the NIR-I and II regions were investigated step by step, from a simple tissue phantom to a three-dimensional cellular system, and further to a tumor-bearing animal model. NIR-II laser exhibited a lower photothermal attenuation coefficient (0.541 at 1064 nm) in a tissue phantom compared with its counterpart (0.959 at 808 nm), which allows it to be more capable of deeptissue PTT in vitro and in vivo. Depth profile analysis elucidated a negative correlation between the microstructural collapse of tumor tissue and the penetration depth. Moreover, the depth-related tumor ablation was also studied by Raman fingerprint analysis, which demonstrated the major biochemical compositional disturbances in photothermal ablated tumor tissues, providing fundamental knowledge to NIR-II deeptissue photothermal therapy.