The uniform and aligned arrangement of tendon cells is a marker of tendon tissue morphology and the embodiment of its biological anisotropy. However, most of the hydrogels used for tendon tissue engineering do not present anisotropic structures. In this work, a magnetically-responsive nanocomposite hydrogel composed of collagen type I (COL I) and aligned iron oxide nanoparticles (IOPs) was investigated for potential application in tendon tissue engineering. COL I with a mixture of remotely aligned IOPs (A/IOPs) and human tendon stem/progenitor cells (COL I-A/IOPs-hTSPCs) was prepared and the alignment of IOPs was induced under a remote magnetic field. Following the gelation of COL I, a stable and anisotropic nanocomposite COL I-A/IOPs hydrogel was formed. In addition, hTSPCs embedded in COL I with random IOPs (COL I-R/IOPs-hTSPCs) and in pure COL I (COL I-hTSPCs) were used as control groups. Cell viability, proliferation, morphology, cell row formation, and alignment of IOPs and hTSPCs were evaluated over time. In addition, a comprehensive gene expression profile of 48 different genes, including tendon-related genes and lineage/cross-linking genes, was obtained by implementing designer quantitative RT-PCR plates. The hTSPCs morphology followed the orientation of the anisotropic COL I-A/IOPs hydrogel with increased row formation in comparison to pristine COL I and COL-R/IOPs. Moreover, higher proliferation rate and significant upregulation of tendon gene markers were measured in comparison to hTSPCs cultivated in the COL I-R/IOPs and COL I. Thus, we suggest that providing the cells with aligned focal contact points, namely the aligned IOPs, is sufficient to provoke an immense effect on the formation of aligned cell rows. Taken together, we report a novel strategy for directing stem cell behavior without the use of exogenous growth factors or pre-aligned COL I fibers, and propose that anisotropic nanocomposite hydrogels hold great potential for tendon tissue engineering applications.