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Cardiac adaptation to hypoxic injury is regulated by dynamic interactions between cardiomyocytes and macrophages, yet the impacts of immune phenotypes on cardiac structure and contractility remain poorly understood. To address this, we developed the immuno-heart on a chip, a novel in vitro platform to investigate cardiomyocyte–macrophage interactions under normoxic and hypoxic conditions. By integrating neonatal rat ventricular myocytes (NRVMs) and bone marrow-derived macrophages—polarized to pro-inflammatory (M1) or pro-healing (M2/M2*) phenotypes—we elucidated the dual protective and detrimental roles macrophages play in modulating cardiomyocyte cytoskeletal architecture and contractility. Pro-inflammatory stimulation reduced cardiomyocyte structural metrics (z-line length, fraction, and integrity) in normoxic co-cultures. Under hypoxia, M1-stimulated NRVM monocultures exhibited declines in cytoskeletal organization—quantified by actin and z-line orientational order parameters. Relative to monocultures, M1-stimulated co-cultures attenuated hypoxia-induced active stress declines but produced weaker normoxic stresses. In contrast, pro-healing stimulation improved normoxic z-line metrics and preserved post-hypoxia cytoskeletal organization but reduced normoxic contractility. Notably, M2-stimulated macrophages restored normoxic contractility and preserved post-hypoxia systolic stress, albeit with increased diastolic stress. RNAseq analysis of M2-stimulated co-cultures identified upregulated structural and immune pathways driving these hypoxia-induced changes. Cytokine profiles revealed stimulation-specific and density-dependent tumor necrosis factor-alpha and interleukin-10 secretion patterns. Together, these findings quantitatively link clinically relevant macrophage phenotypes and cytokines to distinct changes in cardiac structure and contractility, offering mechanistic insights into immune modulation of hypoxia-induced dysfunction. Moreover, the immuno-heart on a chip represents an innovative framework to guide the development of future therapies that integrate immune and cardiac targets to enhance patient outcomes.