Phenylboronic acid (PBA)-based glucose-responsive microneedles that can painlessly deliver insulin and self-regulate the release of insulin according to the blood glucose levels (BGLs) have been considered promising in the treatment of diabetes. However, the reported PBA-based glucose-responsive microneedles are still faced with the challenges of complicated insulin loading process or insulin denaturation. Herein, we report a novel kind of PBA‐based glucose-responsive microneedles derived from PBA‐based glucose-responsive hydrogels. 3-fluoro-4-carboxy-phenylboronic acid-grafted polylysine (PLL-PBA) were synthesized and combined with natural guar gum (GG) to produce PLL-PBA/GG hydrogels via the phenylboronate ester bonds formed between the FCPBA groups on PLL-PBA and the diols on GG. The resulting hydrogels could regulate the release amount of insulin according to the glucose concentrations based on the glucose-induced disintegration mechanism. The PLL-PBA/GG microneedles were prepared via micro-mold casting method using the hydrogels as the precursors. Due to the simple and mild preparation conditions of the microneedles, the bioactivity of insulin was greatly preserved. The microneedles had uniform morphology, moderate skin penetration ability as well as good glucose-responsive insulin release capability. In animal experiments, the PLL-PBA/GG microneedles demonstrated BGLs lowering and maintain performance via glucose-responsive transdermal insulin delivery.