Although genome sequencing technologies have advanced rapidly, microbial genomes still contain numerous genes with unknown functions, posing ongoing challenges for comprehensive genome annotation. Traditional annotation methods are constrained by a lack of scalable experimental techniques and the limitations of conventional homology-based computational approaches. Recent computational innovations, particularly deep learning, have substantially improved gene function prediction, facilitating more efficient annotation of transcription factors, enzymes and other protein classes. Integrating computational and experimental approaches has enabled the development of workflows that systematize gene function discovery, paving the way for faster, more accurate and comprehensive genome annotation. Continued refinement of these integrated methods holds great promise for deepening our understanding of microorganisms. Here we review recent advances in artificial intelligence for gene function discovery and discuss future directions for achieving interpretable and high-throughput artificial intelligence-guided annotation.