Numerous benefits of breastfeeding over infant formula are fully established. The superiority of human milk over bovine milk-based formula is partly due to human milk oligosaccharides (HMOs), a family of over 100 molecules present specifically and substantially in human milk that resemble mucosal glycans. To uncover novel physiological functions and pathways of HMOs, we screened a panel of 165 G-protein coupled receptors (GPCRs) using a blend of 6 HMOs (3'-O-sialyllactose (3'SL), 6'-O-sialyllactose (6'SL), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), 2-O-fucosyllactose (2'FL), and difucosyllactose (diFL)), and followed up positive hits with standard receptor assays. The HMO blend specifically activated GPR35. LNT and 6'SL individually activated GPR35, and they showed synergy when used together. In addition, in vitro fermentation of infant stool samples showed that 2'FL upregulates the production of the GPR35 agonist kynurenic acid (KYNA) by the microbiota. LNT + 6'SL and KYNA showed additive activation of GPR35. Activation by 6'SL and LNT of GPR35, a receptor mediating attenuation of pain and colitis, is to our knowledge the first demonstration of GPCR activation by any HMO. In addition, we demonstrated a remarkable cooperation between nutrition and microbiota towards activation of a host receptor highlighting the close interplay between environment and host-microbe interactions.