In this review, we introduce the activities of endo-β-1,4-galactanases and related enzymes, their biological niches, and biological function. Endo-β-1,4-galactanases are classified in the glycoside hydrolase family 53 in the CAZy database (GH53) and degrade β-1,4-linked galactans and arabinogalactans. These substrates are found as sidechains decorating the backbone of rhamnogalacturonan type I, a “hairy” part of pectin. Pectin is found in the primary cell walls of nonwoody plants and enzymatic degradation of the pectin network is of interest in the production of, e.g., biofuel, fruit juice, and animal feed. β-1,4-Galactanases have also drawn interest due to their importance in the production and metabolism of prebiotic galactooligosaccharides used in formula milk. To date, several structures of endo-β-1,4-galactanases originating from a variety of organisms are known, which highlight structural variance in the length of substrate-binding sites. Based on these structures, we provide here a comprehensive overview on the structure-function relationship within the GH53 family. The acquired structural knowledge has led to engineering of β-1,4-galactanases to alter their biophysical and biochemical features and increase their applicability in industry.