Lanthanide-doped upconverting nanoparticles (Ln-UCNPs) possess optical and physicochemical properties that are promising for the design of new theranostic platforms. This applies in particular to the treatment of cancer. Towards this goal, oleate-capped-NaLuF4:Tm3+(0.5%)/Yb3+(20%)/Gd3+(30%) with an average size of 35 nm ± 2 nm were synthesized by co-precipitation. Due to their hydrophobic surface, these Ln-UCNPs produced agglomerates under cell culture conditions. To assess the cellular response to Ln-UCNPs at the molecular level, we evaluated several key aspects of tumor cell physiology. Using cancer lines of different origins, we demonstrated Ln-UCNP dependent changes of cancer cell biomarkers. Multiple cellular components that regulate tumorigenesis and cancer cell homeostasis were affected. In particular, Ln-UCNPs reduced the abundance of hsp70s, elevated DNA damage, and diminished nucleolin and B23/nucleophosmin, proteins required for the assembly of ribosomes. Treatment with Ln-UCNPs also decreased the concentration of paxillin, a focal adhesion protein that is involved in directed cell migration. Furthermore, epidermal growth factor (EGFR) levels were decreased by Ln-UCNPs for most cancer cell lines examined. Taken together, we identified several potential cancer cell targets that were affected by Ln-UCNPs. Our work thereby provides the foundation to optimize Ln-UCNPs for the targeted killing of tumor cells.