When mice with ad libitum normal chow received diurnal 2-h delivery of highly sweet chow (HSC) containing 65% sucrose for 14 days, their HSC intake in the light phase gradually increased during the HSC delivery session while their normal chow intake in the dark phase significantly decreased. Neural mechanisms underlying feeding entrainment to the diurnal delivery of the palatable food (palatable food-entrainment) remains unclear. Previous studies suggest that food anticipation-related activity in food-deprived rats is mediated by the dorsomedial hypothalamus (DMH) and that the expression of clock genes in the DMH increases or decreases with synchronizing to the timing of food delivery in food-deprived mice. Then, we assumed that the DMH also plays a role in palatable food-entrainment in ad libitum-fed mice. To test the assumption, we examined the effect of selective lesions of the DMH on the palatable food-entrainment in ad libitum-fed mice. The bilateral DMH in male mice (n = 9) was selectively damaged through iontophoretical microinfusion of an excitotoxin, kainic acid, with sparing of the ventromedial hypothalamus locating just ventrally adjacent to the DMH. The other mice (sham; n = 10) received saline instead of the excitotoxin. The basal intakes of normal chow for the corresponding 2-h time period before the HSC delivery session were not different between DMH-lesioned and sham groups. Sham lesioned mice showed 6-7-fold increase of HSC intake compared to their basal intake of normal chow; however, DMH-lesioned mice showed less than 4-fold increase of the HSC intake. The HSC intake in DMH-lesioned mice was significantly smaller than that in the sham group. The nocturnal intake of normal chow in DMH-lesioned mice during the HSC delivery session was significantly greater than that in the sham group. No difference in body weight and preference for sucrose over water in two-bottle test was observed between the groups. These results suggest that the DMH plays a role in the formation, maintenance and/or expression of palatable food-entrainment in ad libitum-fed mice without change of body weight control and taste preference for sweet taste.