Lysine methyltransferase KMT5C catalyzes deposition of trimethylation on histone H4 lysine 20 (H4K20me3), an epigenetic marker usually associated with gene repression and maintenance of heterochromatin. KMT5C is widely expressed in a variety of tissues, however, its functional role in liver has not been explored. Here, we show Kmt5c is a fasting- and glucagon-induced gene in liver which regulates hepatic gluconeogenesis. Loss of KMT5C in hepatocytes results in downregulated gluconeogenic gene expression and compromised glucose output during fasting. KMT5C fosters gluconeogenesis through decreasing ubiquitination-mediated PGC-1α degradation, which is unexpectedly independent of its methyltransferase activity. In fact, KMT5C impedes the E3 ligase RNF34 binding to the C-terminal of PGC-1α and subsequent ubiquitination-associated degradation. The diabetic mice models and patients show elevated KMT5C levels in the livers, and KMT5C knockdown beneficially reduces gluconeogenesis and fasting blood glucose levels. In conclusion, the present study identifies KMT5C as a hepatic gluconeogenesis regulator by affecting PGC-1α stability.