Titanium (Ti) nanorods fabricated using selective corrosion of Ti substrate by anodic technology show better biocompatibility with pre-osteoblast cells. The current study investigated the response of the murine pre-osteoblast cell MC3T3-E1 on Ti nanorod topography and untreated Ti surfaces by means of examination of the morphology and osteogenic differentiation responsible for the pre-osteoblast reaction. The morphology of MC3T3-E1 cells was observed using scanning electron microscopy, and alkaline phosphatase (ALP) activity was measured using a colorimetric assay after incubation for 7, 14, and 21 days. The expression of three osteogenic differentiation markers including ALP, osteocalcin (OCN), and collagen type 1A1 (COL1A1) and two transcription factors including runt related transcription factor 2 (Runx2) and osterix (Osx) at different time points was detected using real-time polymerase chain reaction analysis in both groups. Osx was used to confirm the protein level. The results showed that Ti nanorod surfaces provided prolonged higher levels of ALP activity compared with unmodified Ti surface on the 14th and 21st days. Gene expression analysis of ALP, OCN, and COL1A1 showed significant upregulation with modified nanorod topography after incubation for 14 and 21 days. Osteogenic transcription factors of Runx2 and Osx exhibited changes consistent with the osteogenic differentiation markers, and this may contribute to the persistently active differentiation of MC3T3-E1 cells in the Ti nanorod group. These results demonstrated that the current nanostructured surface may be considered bioadaptive topography to control cellular behaviors and osteoblast differentiation. The in vivo performance and applicability are further required to investigate osseointegration between implant and host bone in the early stages for prevention of aseptic implant loosening.