Background <p>Methanol is a promising renewable C1 feedstock for biomanufacturing. Native methylotrophs such as <i>Pichia pastoris</i> are considered potential chassis strains for methanol utilization. However, the cytotoxicity of methanol limits its usable concentration, posing a major bottleneck in bioproduction. In this study, we aimed to develop a <i>P. pastoris</i> strain with enhanced methanol tolerance and utilization by adaptive laboratory evolution.</p> Results <p>Through serial passaging under increasing methanol concentrations, we obtained an evolved strain that exhibited a specific growth rate of 0.101&#xa0;h^− 1 in a 7% (v/v) methanol, a condition under which the wild-type strain failed to grow. Genome resequencing identified the mutations, and the introduction of individual mutations into the wild-type background demonstrated that mutations in <i>PSR1</i> and <i>BFA1</i> significantly improved growth under methanol stress. Notably, the fastest-growing isolate, designated PM7a, was capable of growing in a minimal medium containing 5% methanol as the sole carbon source, whereas the wild-type strain was unable to do so. Furthermore, the introduction of the β-carotene biosynthesis pathway into both the wild-type and PM7a strains, cultured in a minimal medium containing methanol as the sole carbon source, resulted in a 4.91-fold higher titer in PM7a.</p> Conclusions <p>Taken together, our findings demonstrate that ALE facilitates the development of strains with enhanced methanol tolerance, growth, and biosynthetic performance. These findings highlight the potential of the evolved <i>P. pastoris</i> strain as a robust chassis for methanol-based biomanufacturing.</p>