Left-behind children (LBC) are a unique population in China, whose numbers have increased dramatically in recent years. Most caregivers of left-behind children (CLBC) are grandparents who lack knowledge about proper nutrition and food practice, putting LBC at greater risk for malnutrition. A cluster randomized controlled trial was carried to assess the effectiveness of the conditional cash transfer (CCT) program. Forty rural villages from Fenghuang County and Pingjiang County of Hunan province were selected. The villages were randomized into the intervention and control groups. In the intervention group, caregivers received a cash transfer conditional on bimonthly health education attendance, bringing LBC in for vaccinations, and on-time annual health checks. The control group received routine health services only. Two rounds of questionnaire surveys were conducted in March 2015 and July 2016. Questionnaires and in-person interviews were used to assess the changes in nutritional knowledge and food practices among CLBC. Among 447 valid subjects, CLBC in the intervention group were significantly more likely to correctly understand the importance of children’s height and weight measurements, food variety, inclusion of eggs and dairy in the diet, and anemia identification and prevention. Intervention group CLBC were also significantly more likely to prepare dairy products and eggs for their children. Generalized liner mixed model (GLMM) analysis showed that CLBC nutrition knowledge was improved significantly in the intervention group (adjusted p value = 0.01), and there were also positive changes in their food practice (adjusted p value = 0.047). This CCT intervention turned to be effective with respect to rural caregivers’ nutritional knowledge and food practice behavior. The findings from this project could be helpful for future health strategies targeting rural children, in particular the LBC group.