Electrochemically engineered titania (TiO₂) nanopores enable tailored cellular function; however, the cellular mechanosensing mechanisms dictating the cell response and soft tissue integration are yet to be elucidated. Here, we report the fabrication of anisotropic TiO₂ nanopores with diameters of 46 and 66 nm on microrough titanium (Ti) via electrochemical anodization, towards short- and long-term guidance of human primary gingival fibroblasts (hGFs). Cells on tissue culture plates and bare Ti substrates were used as controls. Notably, we show that nanopores with a diameter of 66 nm induced more mature focal adhesions of vinculin and paxillin at the membrane, encouraged the development of actin fibers at focal adhesion sites, and led to elongated cell and nuclear shape. These topographical-driven changes were attributed to the Ras-related C3 botulinum toxin substrate 1 (Rac 1) GTPase pathway and nuclear localisation of LAMIN A/C and yes-associated protein (YAP) and associated with increased ligament differentiation with elevated expression of the ligament marker Mohawk homeobox (MKX). Study findings reveal that minor tuning of nanopore diameter is a powerful tool to explore intracellular and nuclear mechanotransduction and gain insight into the relationships between nanomaterials and mechanoresponsive cellular elements.