As an excellent source of abundant and renewable industrial and agricultural raw materials, the Moso bamboo stem has excellent mechanical properties, which are closely related to the cell wall composition and structure. However, the molecular regulation of cell wall biosynthesis (CWB) in Moso bamboo remains unclear. We used Moso bamboo protoplasts to investigate CWB. Transcriptome and proteome of embryogenic calli and protoplasts at four time-points (0, 24, 72, and 120 h) were comprehensively analyzed to clarify the transcriptional and translational changes mediating CWB. In total, 41,014 unigenes and 7,454 proteins were identified. There were significant differences between the differentially expressed genes and differentially abundant proteins regarding the enriched GO terms and KEGG pathways, reflecting the temporal and spatial specificities of the samples at the transcript and protein levels. Transcriptomic and proteomic analyses indicated that protoplast cell wall regeneration requires the regulation of gene expression, the guidance of the cytoskeleton system, and sufficient base materials. Furthermore, the new cell wall is more acetylated than methylated, and it may contain a small amount of lignin. Overall, we provide new insights into Moso bamboo protoplast cell wall reconstruction that may be relevant for further characterizing the molecular mechanism controlling CWB in monocotyledons.