Smoking is identified as a risk factor for periodontitis. Carbon monoxide (CO)-releasing molecule-3 (CORM-3) is a compound that has demonstrated anti-inflammatory effects in vitro and in vivo studies. The present study aimed to investigate the effects of CORM-3 on the expression of inflammatory and osteoclastogenic cytokines in human periodontal ligament cells (PDLCs) stimulated by nicotine and lipopolysaccharide (LPS). The cells were pretreated with CORM-3 and then cultured in medium in the presence of nicotine and LPS. The mRNA and protein expression levels of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX‑2), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and heme oxygenase-1 (HO-1) were evaluated using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of these cytokines were also evaluated in PDLCs transiently transfected with HO-1 small interfering RNA (siRNA) in response to nicotine and LPS stimulation. CORM-3 attenuated the LPS- and nicotine-induced production of PGE2, COX-2 and RANKL in human PDLCs by releasing CO, and upregulated the expression of OPG. However, these effects of CORM-3 were abrogated when HO-1 siRNA was transiently transfected into the cells. These results demonstrate that CORM-3 exerts anti-inflammatory and anti-osteoclastogenic effects on nicotine- and LPS-stimulated human PDLCs via the HO-1 pathway, which suggests its promising potential for use in the treatment of inflammatory periodontal disease.