Purpose: Traditional two-dimensional (2D) in vitro models of human skeletal muscle are limited in their ability to fully mimic in vivo muscle, as in vivo muscle exists in a complex three-dimensional (3D) structure. We have developed a novel engineered three-dimensional (3D) myobundle in vitro model that we believe more closely models skeletal muscle behavior. Here, we determined baseline gene expression differences among three models: the 3D myobundles, 2D cell cultures, and explant biopsies. Methods: Previously collected skeletal muscle (vastus lateralis) biopsy samples from adult men and women (n = 6) were used. Each sample was used to generate the following groups: explant (RNA from biopsy), 2D (RNA from differentiated myotubes) and 3D (RNA from 3D myobundles seeded from each primary sample). 200ng of isolated RNA for each sample was used to generate global gene expression profiles (HumanHT-12 v4.0 Gene Expression BeadChip Arrays). Data were processed using Illumina Genome Studio and imported into Partek Genomics Suite for statistical analysis. Differential gene expression was assessed via 2-way ANOVA (group*ID) with the following post-hoc comparisons: 2D/biopsy, 3D/biopsy and 3D/2D. Resultant lists were filtered at p<0.01 and fold change >|1.5|. Biological pathway analyses of differentially regulated gene sets were done using Ingenuity Pathway Analysis. Results: ANOVA detected 3754 genes different between 2D/biopsy, 3273 genes different between 3D/biopsy and 488 genes different between 3D/2D cultures. Biological pathway analysis identified representation of the following canonical pathways in our gene set: calcium signaling (26 genes; z-score=1.508, -log p-value=13.9), integrin signaling (28 genes; z-score=-2.711, -log p-value=13.5), and actin cytoskeleton signaling (29 genes; -log p-value=12.3). Conclusion: The 3D cell myobundle system produced relatively fewer differences from biopsies as compared to 2D cell cultures, but some significant differences from biopsy samples remain. Comparison of 3D to 2D culture systems shows transcriptional changes that align with increases in calcium signaling, while downregulations in the actin cytoskeleton and integrin signaling demonstrate significant structural differences between the two in vitro models tested.