This study clarifies how hydroxyapatite (HA) allocation and microgroove dimension affect mouse preosteoblastic MC3T3-E1 cell functions on microgrooved substrates of polymer nanocomposites. Using replica molding from micromachined silicon wafer templates, we fabricated photocured poly(ε-caprolactone) triacrylate (PCLTA)/HA nanocomposite substrates with parallel microgrooves (two groove widths of 5 and 15 μm and one groove depth of 5 μm). Four types of microgrooved substrates were made: "homogeneous" ones of PCLTA and PCLTA/HA with uniform distribution and two "heterogeneous" laminated microgrooved substrates with HA only in the PCLTA matrix in the ridges or bottom. These substrates were used to regulate MC3T3-E1 cell attachment, proliferation, alignment, nuclear circularity and distribution, and mineralization. MC3T3-E1 cell attachment and proliferation were much higher on the microgrooved substrates of PCLTA/HA than on those of PCLTA, in particular, on the 5 μm wide microgrooved substrate with PCLTA/HA ridges and PCLTA bottom. The shape and distribution of MC3T3-E1 cytoskeleton and nuclei were altered by the substrate topography and HA allocation. For 5 μm wide heterogeneous microgrooved substrates with HA only in the ridges, MC3T3-E1 cells exhibited better spreading perpendicular to the microgrooves but tended to extend along the microgrooves containing HA in the bottom. The widest cells and the roundest/largest cell nuclei were observed on the heterogeneous substrate with PCLTA/HA ridges, while the narrowest cells with the best elongation were found on the homogeneous PCLTA/HA substrate. The trend in MC3T3-E1 cell mineralization on the substrates was consistent with that in cell/nuclear elongation. Osteocalcin mRNA expression was significantly higher on the PCLTA/HA substrates than on the PCLTA ones and also on the microgrooved substrates of PCLTA/HA than on the flat ones, regardless of the groove width of 5 or 15 μm.