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Storage proteins are essential for seed germination and seedling growth, as they provide an indispensable nitrogen source and energy. Our previous report highlighted the defective endosperm development in the <i>serine hydroxymethyltransferase</i><i> 4</i> (<i>OsSHMT4</i>) gene mutant, <i>floury endosperm20</i>-<i>1</i> (<i>flo20-1</i>). However, the alterations in storage protein content and distribution within the <i>flo20-1</i> endosperm remained unclear. Here, the immunocytochemistry analyses revealed a deficiency in storage protein accumulation in <i>flo20-1</i>. Electron microscopic observation uncovered abnormal morphological structures in protein bodies (PBI and PBII) in <i>flo20-1</i>. Immunofluorescence labeling demonstrated that aberrant prolamin composition could lead to the subsequent formation and deposition of atypical structures in protein body I (PBI), and decreased levels of glutelins and globulin resulted in protein body II (PBII) malformation. Further RNA-seq data combined with qRT-PCR results indicated that altered transcription levels of storage protein structural genes were responsible for the abnormal synthesis and accumulation of storage protein, which further led to non-concentric ring structural PBIs and amorphous PBIIs. Collectively, our findings further underscored that <i>OsSHMT4</i> is required for the synthesis and accumulation of storage proteins and storage organelle formation in endosperm cells.