This work presents an auxetic hexachiral cantilever substrate for low-frequency vibration energy harvesting applications. Auxetics are materials with negative Poisson’s ratio that develop stresses of the same nature under mechanical loading, which can be advantageously used in designing energy harvesters with enhanced power output. The proposed harvester is fabricated by attaching a piezo patch on a 3D printed polylactic acid (PLA) hexachiral substrate to convert the mechanical response to electrical output. Experiments are conducted to characterize the vibration and electrical properties of the harvester. A 3D finite element (FE) model is developed and validated with experimental voltage obtained for different electrical resistance. As the first mode generates maximum power, an equivalent single degree of freedom (SDOF) semi-analytical model is formulated and validated with experiments and FE results. The proposed harvester has a natural frequency of 23 Hz with a voltage output of 9.1 V at 250 kΩ. The developed models are used to study the influence of hexachiral geometry, electrical and mechanical loading on the electro-mechanical response. The harvester voltage is influenced by the ligament thickness and is found to increase linearly with an increase in mechanical loading. Further, the enhancement in performance by the addition of hexachiral sub patch to a plain beam is investigated, followed by a comparison with harvesters having plain, hexagonal and re-entrant geometries. The results show that the hexachiral harvester has the lowest first mode frequency with a power output about 20 and 3 times the plain and re-entrant harvesters, respectively. Finally, random vibration analysis of the hexachiral harvester is carried out to evaluate its performance under ambient loading, and the results show that the semi-analytical model is a computationally efficient alternative to study the first mode behavior. The findings of the study demonstrate the potential of the proposed hexachiral harvester for low-frequency applications.