Gold nanoparticles (AuNPs) have been extensively studied for photothermal cancer therapy because AuNPs can generate heat upon near-infrared irradiation. However, improving their tumor-targeting efficiency and optimizing the nanoparticle size for maximizing the photothermal effect remain challenging. We demonstrate that mesenchymal stem cells (MSCs) can aggregate pH-sensitive gold nanoparticles (PSAuNPs) in mildly acidic endosomes, target tumors, and be used for photothermal therapy. These aggregated structures had a higher cellular retention in comparison to pH-insensitive, control AuNPs (cAuNPs), which is important for the cell-based delivery process. PSAuNP-laden MSCs (MSC-PSAuNPs) injected intravenously to tumor-bearing mice show a 37-fold higher tumor-targeting efficiency (5.6% of the injected dose) and 8.3 °C higher heat generation compared to injections of cAuNPs after irradiation, which results in a significantly enhanced anticancer effect.