Background: Although limited work has been done on investigating the effects of Histone deacetylases (HDACs) on osteoclast formation and function, HDACs have been shown to regulate gene expression and may work as a critical regulator of osteoclast differentiation, especially class II and IV HDACs which function as inhibitors. The study aims to investigate whether HDAC4 has an effect on the compressive force (CF)-induced osteoclast differentiation in human periodontal ligament cells (PDLCs) and its role in regulating the mitogen-activated protein kinase/beta-catenin (MAPK/β-catenin) signaling pathway. Methods: The PDLCs were isolated from normal premolar extracted for orthodontic purposes. The expression of HDAC4 during osteoclast differentiation was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Then, the characteristic changes of PDLCs were evaluated utilizing tartrate-resistant acid phosphatase (TRAP) staining, cell counting kit-8 (CCK-8), and colony-forming units-fibroblast (CFU-F) procedures. Then, the CF group was transfected and divided into four groups: CF cells transfected with small interference RNA (Si-RNA) against HDAC4 (Si-HDAC4) and the corresponding scramble Si-RNA as negative control (CF cells transfected with scramble small interfering (Si)-RNA, Si-NC), cells transfected with lentiviral vectors encoding HDAC4 overexpression-RNA (CF cells transfected with lentiviral vectors encoding HDAC4 overexpression-RNA, OE-HDAC4) and the corresponding blank vectors as negative control (CF cells transfected with blank lentiviral vectors, OE-NC). The expression of MAPK/β-catenin pathway-related proteins in these groups was detected, and the regulation of HDAC4 on the MAPK/β-catenin pathway was verified by adding pathway activator isomycin or cells transfected with Si-β-catenin. Results: It was observed that the expression of HDAC4 decreased in PDLCs after CF. Inhibition of HDAC4 promoted osteoclast differentiation in PDLCs. The study also revealed that due to the inhibition of HDAC4, the relative expression of p-p38/p38 in PDLCs was significantly down-regulated (p < 0.001). However, using the MAPK pathway activator, the osteoclast differentiation expression level returned to or above the normal levels (p < 0.05). Additionally, we investigated that the inhibition of HDAC4 significantly upregulated the relative expression level of β-catenin (p < 0.001). Moreover, after transfecting the cells with Si-β-catenin, the progress of osteoclast differentiation was restored to or above the normal level. Conclusion: It is concluded that suppressing HDAC4 expression could promote osteoclast differentiation in human PDLCs. HDAC4 also plays an important role in regulating the MAPK/β-Catenin signaling pathway. At present, there are a few applications of orthodontic tooth movement (OTM) treatment using PDLCs, this study could aid the clinical applications of PDLCs for OTM treatment.