DMSO is one of the most common solvents used experimentally to dissolve hydrophobic substances for in vivo and in vitro purposes. A wide range of pharmacological effects exerted by DMSO has been documented in both animal and human experimental models. However, only a few and sometimes contrasting data about the effects of DMSO in animal models of nociception and inflammation are presently available. In this study, we evaluated the effects induced by DMSO and a DMSO-containing saline on thermal and chemical nociception, inflammation and locomotor activity in CD1 mice. We demonstrated that centrally or orally administered DMSO displayed anti-nociceptive effects to thermal (hot plate and tail-flick test) and chemical (formalin test) stimuli. Conversely, DMSO was able to increase both nociceptive phases in the formalin test when applied subcutaneously in the dorsal surface of the mouse hind paws 10 min before formalin administration. Oral administration of DMSO produced anti-inflammatory effects on zymosan-induced edema in the mouse paw, whereas local administration potentiated the inflammatory action exerted by zymosan. Oral and central, but not local, administration of DMSO improved the mouse locomotor activity. These results suggest that DMSO displayed opposite effects on nociception and inflammation, depending on the route of administration. New and helpful evidence about DMSO laboratory applications need to be considered in the in vivo studies to assess correctly the pharmacological properties of investigated drugs.