Galectins are widely expressed galactose‐binding lectins implied, for example, in immune regulation, metastatic spreading, and pathogen recognition. N‐Acetyllactosamine (Galβ1‐4GlcNAc, LacNAc) and its oligomeric or glycosylated forms are natural ligands of galectins. To probe substrate specificity and binding mode of galectins, we synthesized a complete series of six mono‐deoxyfluorinated analogues of LacNAc, in which each hydroxyl has been selectively replaced by fluorine while the anomeric position has been protected as methyl β‐glycoside. Initial evaluation of their binding to human galectin‐1 and ‐3 by ELISA and 19F NMR T2‐filter revealed that deoxyfluorination at C3, C4′ and C6′ completely abolished binding to galectin‐1 but very weak binding to galectin‐3 was still detectable. Moreover, deoxyfluorination of C2′ caused an approximately 8‐fold increase in the binding affinity towards galectin‐1, whereas binding to galectin‐3 was essentially not affected. Lipophilicity measurement revealed that deoxyfluorination at the Gal moiety affects log P very differently compared to deoxyfluorination at the GlcNAc moiety.