[Objectives] The present study aims to test the effect of maternal care level on offspring boldness of Labrador Retrievers. [Methods] Behaviors of seven litters of Labrador Retrievers (total of 54 puppies) and their mothers living in standardized rearing conditions in China Guide Dog Training Centre (Dalian) were studied. Maternal care behavior of seven female dogs was recorded continuously from day 1 to 21 after birth with a surveillance camera, and video files were stored for later scoring and analysis. 90 min (comprised of 6 different periods of 15 min, selected from 7:00﹣8:00, 9:00﹣10:00, 12:00﹣13:00, 15:00﹣16:00, 18:00﹣19:00, and 20:00﹣21:00) video of each day was scoring for maternal behavior variables. The variables recorded were:contact with the puppy, stay in the nursing box, nursing the puppy, and licking the puppy. A principal component analysis (PCA) was performed on the four variables and PC-scores were assigned to each mother. To assess the boldness of puppies, each puppy was subjected puppy tests consisting of passive test, metallic sound test, umbrella test, toy test, ramp and tunnel test at 6﹣8 weeks of age (Fig. 1). The Mann-Whitney U test was conducted to contrast the boldness scores of puppies between high PC-scores group and low PC-scores group, and between genders. [Results] The principal component analysis of the maternal care data revealed that just one factor with eigenvalues greater than one (Table 2). The puppies were divided into two groups based on PC-scores of female dogs:high level of maternal care (n = 24) and low level of maternal care (n = 30) (Table 3). Pearson correlation analysis was used to analyze the scores of 13 behavioral variables in the puppy test by two raters, and the inter-rater reliability was higher than 0.8 (Table 4). Mann-Whitney U test results showed that there was no significant difference (P > 0.05) in scores of other behavioral variables between male and female puppies except for the startle of metallic sound test and tension of metallic sound test. 4 (Table 5), and different maternal levels had significant impacts on 9 behavioral variable scores out of 13 (P < 0.05) (Fig. 2). Among them, the puppies with low level of maternal care had lower tension scores in all the puppy sub-tests than those with high level of maternal care. [Conclusion] In conclusion, low maternal care brings intermittent and appropriate intensity of early life stress to puppies, which makes them show better adaptability and greater boldness when facing new environmental stimulation. There is a new suggestion that breeding dogs with low levels of maternal care have a better influence on the boldness development of offspring. However, how to define the level of maternal care to ensure the physically healthy development of puppies while providing moderate early stress to promote the development of puppies’ boldness still needs more research.