Strongyloides stercoralis infection is caused by skin penetration of third-stage larvae (L3s). We studied skin penetration of L3s of Strongyloides ratti using an in vitro assay that has been used previously to study Angiostrongylus cantonensis, an agarose membrane with a temperature gradient, and scanning electron microscopy. Our results revealed that skin penetration of L3s depended on host skin temperature. When the target temperature of the outer liquid was 37°C, more than 80% of L3s penetrated the skin, but penetration was only 60% when the target temperature was 20°C. Thirdstage larvae moved rapidly on the agarose membrane toward optimum temperature area for this parasite, which indicates that L3 has a sensor that is sensitive to temperature changes. Penetration rate for hosts such as cat (36%), dog (32%), and bird (13%) were significantly lower than that for rat (82%). Although we could not establish the reason, L3s seemed to have an ability to differentiate these hosts at the time of penetration. By using scanning electron microscopy, penetration of L3s could be observed within 10 min. We demonstrated thermotaxis of L3 of S. ratti, and this peculiar characteristic seemed to have a close relationship with the process of searching for the host.