Objective@#To investigate the relationship between human bocavirus 2 (HBoV2) infection and acute diarrhea in children younger than 5 years of age in a case-control study.@*Methods@#This was a prospective case-control study. During May 2016 to December 2016, fecal specimens were collected from children ≤5 years of age with acute diarrhea who visited the Affiliated Children's Hospital of Capital Institute of Pediatrics (case group), or from children ≤5 years of age without diarrhea from Longtan Community Medical Service Center, Beijing (control group). The case group (n=240) and the control group (n=240) were divided into 8 age subgroups: ≤1 month old, >1-3 months old, >3-6 months old, >6-12 months old,>1-2 years old,>2-3 years old,>3-4 years old and >4-5 years old, and there were 30 cases in each age subgroup. The specimens were tested for 7 types of diarrhea-associated viruses, especially for HBoV2 by real-time PCR method. The HBoV2 viral load was predicted according to the cycle threshold (Ct). Finally, t-test was used to compare the differences between groups.@*Results@#In the case group (n=240), the positive rate of norovirus was 16.7% (40 cases); rotavirus, 10.8% (26 cases); HBoV2, 7.5% (18 cases); adenovirus, 7.1% (17 cases); astrovirus, 6.3% (15 cases); parachovirus, 3.8% (9 cases); and Aich virus, 0.4% (1 case). The positive rates of HBoV2 in case group (7.5%, 18 cases) and control group (5.0%, 12 cases) showed no significant difference (χ2=1.280, P=0.258), as well as in different age groups (all P>0.05) . However, the mean viral load of the HBoV2 in the case group (1×109copies/L with cycle threshold (Ct) 25.8) was higher than that of control group (1×105copies/L with Ct 33.8), showing a significant difference (t=0.597, P=0.000).@*Conclusions@#Norovirus and rotavirus are still the important viral pathogens in children with acute diarrhea. A higher load of HBoV2 may indicate a higher risk of acute diarrhea in children ≤5 years of age in Beijing.