Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, phagocytic, pro-healing phenotype, whereas OPN inhibition triggered a pro-inflammatory phenotype. Here, we applied a global proteome profiling via mass spectrometry analysis to gain a mechanistic understanding of OPN suppression versus induction in macrophages. We identified over 630 differentially expressed proteins (DEPs) in OPN knockout (OPNKO) or GA-stimulated versus wild type (WT) macrophages. Two topmost downregulated DEPs in OPN-deficient macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We confirmed UCHL1 expression in BMMΦ, which was previously recognized as a neuronal-specific protein. Further, immunoprecipitation assays suggest a direct interaction of OPN and UCHL1 proteins. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (ROS, Lamp1/2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, Western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis (proteostasis) in macrophages—inducing apoptosis and inhibiting translation—whereas GA-stimulated OPN induction restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostasis via regulation of cell viability, UCHL1-UPS, and protein synthesis, indicating its potential application in immunotherapy.