The Pal/Rim pathway essential for fungal adaptation to ambient pH has been unexplored in Beauveria bassiana, a classic fungal entomopathogen. Here, we show the characterized Pal pathway comprising transcription factor PacC and upstream six Pal partners (PalA/B/C/F/H/I) in B. bassiana. Their coding genes were all transcribed most abundantly in standard wild-type culture under the alkaline condition of pH 9. Deletion of pacC or each pal gene resulted in a significant delay of culture acidification in a minimal broth (initial pH = 7.3). This delay concurred with altered accumulation levels of intra/extracellular organic acids and drastically depressed expression of some enzyme genes required for the syntheses of oxalic and lactic acids. Our deletion mutants except Delta palI showed growth defects and maximal sensitivity to NaCl, KCl, LiCl, or sorbitol at pH 9, an alkaline condition leading to fragmented vacuoles in their hyphal cells exposed to osmotic stress. In these mutants, conidiation was significantly facilitated at pH 3 more than at pH 7 but suppressed slightly at pH 9. Mild virulence defects also occurred in the absence of pacC or any pal gene. These changes were restored by targeted gene complementation. Taken together, PacC and Pal partners regulate the growth, conidiation, and osmotolerance of B. bassiana in a pH-dependent manner, highlighting their vitality for the fungal pH response.