Different and selective vulnerability among motor neuron subtypes are a fundamental, but unexplained, feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) motor neurons are the most vulnerable, and fast fatigue-resistant/slow (FR/S) motor neurons are relatively resistant. We identified that osteopontin (OPN) can serve as a marker of FR/S motor neurons, whereas matrix metalloproteinase-9 (MMP9) is expressed by FF motor neurons in mice. In SOD1G93A ALS model mice, as the disease progressed, OPN was secreted and accumulated as granular deposits in the extracellular matrix. We also detected OPN/MMP9 co-expressed motor neurons around the disease onset. These double positive motor neurons showed the expression of αvβ3 integrin (OPN receptor) and up-regulation of ER stress markers. We discovered that the double positive motor neurons are remodeled FR/S motor neurons, which compensated for FF motor neuron degeneration (the first wave of degeneration). Genetic ablation of OPN delayed the onset of disease, but later accelerated disease progression. This reflects two modes of OPN involvement in the pathogenesis of ALS: cell-autonomous and non-cell-autonomous effects on motor neuron vulnerability. Our study suggests that OPN expressed in FR/S motor neurons is involved in the second wave of motor neuron degeneration in ALS, and an OPN-αvβ3 integrin-MMP9 axis could be a potentially useful therapeutic target for ALS.