Histone deacetylases (HDACs) are enzymes that play crucial roles in cellular processes by hydrolyzing acetyl-L-lysine side chains in core histones, thereby regulating gene expression and maintaining homeostasis. Histone deacetylase inhibitors (HDACi) have emerged as promising agents, particularly in cancer treatment, due to their ability to induce cytotoxic and pro-apoptotic effects. Selective HDAC6 inhibitors, such as ITF3756, have shown low off-target toxicity and promising pharmacological activities, but their poor water solubility limits their application in nanoparticulate drug delivery systems. Here, we optimized a nanostructured lipid carrier (NLC) formulation for delivering ITF3756 using the design of experiments (DOE) and response surface methodology (RSM). An interaction between the factor surfactant and formulation volume was observed, thus demonstrating that the surfactant concentration impacts the NLC size. It can be speculated that the higher the amount of the drug in the formulation, the lower the polydispersion index (PDI), thus resulting in more stable nanostructures. The optimized ITF3756-NLC demonstrated a size of 51.1 ± 0.3 nm, 8.85 ± 4.71 mV charge, and high entrapment efficiency (EE%), maintaining stability for 60 days. Moreover, ITF3756-NLC enhancedα-tubulin acetylation in melanoma, lung, and brain cancer cell lines, indicating retained or improved bioactivity. The ITF3756-NLC formulation offers a viable approach for enhancing the bioavailability and therapeutic efficacy of HDAC6 inhibitors, demonstrating potential for clinical applications in cancer immunotherapy.