Both intrinsic material properties and topographical features are critical in influencing cell-biomaterial interactions. We present a systematic investigation of regulating mouse pre-osteoblastic MC3T3-E1 cell behavior on biodegradable polymer substrates with distinct mechanical properties and concentric microgrooves. The precursors for fabricating substrates used here were two poly(ϵ-caprolactone) triacrylates (PCLTAs) synthesized from poly(ϵ-caprolactone) triols with molecular weights of ∼7000 and ∼10000 g mol−1. These two PCLTAs were photo-crosslinked into PCL networks with distinct thermal, rheological, and mechanical properties at physiological temperature because of their different crystallinities and melting temperatures. Microgrooved substrates with four groove widths of 7.5, 16.1, 44.2, and 91.2 μm and three groove depths of 0.2, 1, and 10 μm were prepared through replica molding, i.e., photo-crosslinking PCLTA on micro-fabricated silicon wafers with pre-designed concentric groove patterns. MC3T3-E1 cell attachment and proliferation could be better supported by the stiffer substrates while not significantly influenced by the microgrooves. Microgroove dimensions could regulate MC3T3-E1 cell alignment, nuclear shape and distribution, mineralization, and gene expression. Among the microgrooves with a fixed depth of 10 μm, the smallest width of 7.5 μm could align and elongate the cytoskeleton and nuclei most efficiently. Strikingly, higher mineral deposition and upregulation of osteocalcin gene expression were found in the narrower microgrooves when the groove depth was 10 μm.