Titanium dioxide nanoparticles (TiO NPs) represent a scientific breakthrough in the areas of biological and medicinal applications. Interaction of TiO NPs with components of innate immune system remains elusive. This study explored in vitro immunotoxicity of murine macrophage RAW 264.7 to TiO NPs (20 nm, negative charge) and its underlying molecular mechanism by way of immunoredox profiling. In this study, chemically synthesized BSA-functionalized TiO NPs (20 nm, negative charge) were characterized and immunotoxicity was investigated on RAW 264.7 cells. We found that reactive oxygen species levels significantly increased with increasing nitric oxide production, whereas depleting endogenous antioxidant super oxide dismutase as well as nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels. Furthermore, NPs exposure increased the expression of apoptotic factors such as BAX, BIM, and PUMA with disruption of mitochondrial membrane potential (Δψ ) that lead to decrease in immunocytes. Molecular immune profiling revealed the activation of multiple toll-like receptors (TLRs) 4/9/12/13 simultaneously with the phosphorylation of p-p38MAPK and p-SAPK/c-Jun N-terminal kinase (JNK) compared to untreated control. Collectively, this study shows that the molecular nature of TiO NP-induced immunotoxicity in RAW 264.7 macrophage is simultaneous induction of immunocyte apoptosis and multiple TLRs signaling through oxidative stress-dependent SAPK/JNK and p38 mitogen-associated protein kinase activation. This is the first study to address newer molecular mechanism of TiO NP-induced immunotoxicity.