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The Lim1 transcription factor is required in Drosophila for patterning the eye-antennal disk. At the adult stage, Lim1 is strongly expressed in Johnston’s Organ (JO) neurons, the antennal auditory organ. Using RNAi-mediated knockdown of Lim1 using a strong neuronal driver, we find a significant reduction in electrophysiological responses to auditory stimuli, recorded from the antennal nerve. This reduction can be accounted for by Lim1 knockdown in the auditory subset of JO neurons, with no effect of knockdown in JO neuron subsets associated with wind or gravity detection. Conversely, Lim1 knockdown in JO sense organ precursors had no effect on hearing. Mosaic animals with antennal clones of the Lim1^E9 null mutation showed morphological defects in the antenna, and significant auditory electrophysiological defects. Our results are consistent with two distinct functions for Lim1 in the antenna, including an early patterning function in the eye-antennal disk, and a later neural differentiation function in the JO neurons.