The role of natural variations in pre-weaning litter size in rodent adult emotionality and the importance of maternal care as a possible mediating factor have been frequently neglected. To address these issues, maternal behaviour of Sprague-Dawley dams differing in natural number of pups was studied for the first seven postnatal days. Later, adult behaviour of representative male offspring was studied in the elevated plus-maze, the circular corridor, the dark-light box and the forced swimming test. Three groups of offspring were selected in function of the number of littermates: L<10 group (less than 10 pups per dam), L10-15 (between 10 and 15 pups per dam) and L>15 group (more than 15 pups per dam). L<10 litters showed a reduced habituation of activity across time in a circular corridor and as compared to L>15 litters, L<10 litters showed a lower activity during the first 5 min of exposure to the circular corridor. L<10 litters had also higher signs of anxiety in the elevated plus-maze, in comparison to the other two groups. In addition, L<10 litters showed in the forced swimming test reduced struggling and more mild swimming behavior than the other two groups. These abnormalities in L<10 litters are not explained by maternal behavior since they received individually more maternal care than L>15, as assessed by total licking-grooming observed during the whole observation period divided by number of pups. Although previous data from several laboratories have demonstrated that low maternal care is associated with heightened emotionality at adulthood, the present results suggest an important contribution of spontaneous litter size to adult emotional behavior that cannot be explained by concomitant changes in maternal care.