The objective of the present study was to demonstrate the anti-inflammatory and antinociceptive properties of agaricoglycerides of the fermented mushroom of Grifola frondosa (AGF). The effects of AGF on interleukin-1β (IL-1β) levels, tumor necrosis factor-α (TNF-α) levels, nuclear factor kappa B (NF-κB) levels, intercellular adhesion molecule-1 (ICAM-1) levels, cyclooxygenase-2 (COX-2) levels, and inducible nitric oxide synthase (iNOS) levels were determined by ELISA. The antinociceptive effects of AGF were also analyzed in acetic acid-induced pain model and formalin-induced inflammatory pain model, respectively. At the same time, the pharmacokinetic assay of AGF was also made. AGF at the dose level of 500 mg/kg significantly inhibited LPS-induced upregulation of NF-κB activation and the production of IL-1β, TNF-α, iNOS, ICAM-1, and COX-2. Moreover, AGF at the dose level of 500 mg/kg suppressed the acetic acid-induced abdominal constrictions (p < 0.05) and the formalin-induced spontaneous nociceptive behaviors (p < 0.05) in rats. The total plasma concentrations of drug after oral administration of AGF at the dose level of 500 mg/kg led to an improvement in oral bioavailability. It accounts for the anti-inflammatory and antinociceptive activity of AGF. The present study demonstrated that AGF at the dose level of 500 mg/kg has important anti-inflammatory and antinociceptive effects in preclinical models of inflammation and in some models of pain and thus may be used as an alternative medicine for inflammatory pain.