Objective To investigate the role of mesencephalic astrocyte-derived neurotrophic factor (MANF) in the browning of white adipose tissue in mice. Methods Adult male wild-type mice were divided into two groups and housed at room temperature or 4°C for 5 days, respectively. After confirming the successful establishment of the white adipose tissue (WAT） browning model using HE staining, immunohistochemical staining, Western blotting, and Real-time PCR, the expression levels of MANF were detected at both protein and mRNA levels. Adeno-associated virus (AAV)-mediated local injection was used to establish the control group (AAV-EGFP) and the experimental group (AAV-MANF). The expression levels of tyrosine hydroxylase (TH), thermogenic genes, and mitochondrial-related genes were detected by immunohistochemical staining, Western blotting, and Real-time PCR. Results Compared with mice housed at room temperature, mice in the cold stimulation group showed smaller cell sizes in inguinal adipose tissue and epididymal adipose tissue，enhanced uncoupling protein 1 (UCP1) immunostaining，upregulated UCP1 expression at both protein and mRNA levels, increased expression of thermogenic genes and mitochondrial-related genes, elevated TH protein level, as well as upregulated MANF expression at both protein and mRNA levels. After local overexpression of MANF in inguinal adipose tissue of mice, the inguinal adipocyte size was reduced, the number of UCP1-positive cells was increased, the expression of UCP1 was upregulated at both protein and mRNA levels, the TH protein level was elevated, and the expression of thermogenic genes and mitochondrial genes was also increased. Conclusion The upregulation of MANF expression is closely associated with WAT browning, and local overexpression of MANF in inguinal adipose tissue of mice can promote the browning of WAT.