The mitogen-activated protein kinase (MAPK) kinase Ste7 has a conserved Ser/Thr loop (S/T-X-4(6)-S/T) that can activate the MAPK Fus3 or Kss1 for the regulation of pheromone response and filamentous growth in model yeast. Here, we show that not only the loop but also four C-terminal Ser/Thr residues are essential for Ste7 to function in the Fus3 cascade of Beauveria bassiana, a filamentous fungal insect pathogen. Mutagenesis of either looped S216/T220 or C-terminal S362 resulted in the same severe defects in conidial germination, hyphal growth, aerial conidiation, and submerged blastospore production as the ste7 deletion, followed by a complete loss of virulence and similarly increased cell sensitivities to osmotic salts, oxidants, heat shock and UV-B irradiation. Mutagenesis of three other Ser/Thr residues (S391, S440, and T485) also caused severe defects in most of the mentioned phenotypes. These defects correlated well with dramatically reduced transcript levels of some phenotype-related genes. These genes encode a transcription factor (CreA) essential for carbon/nitrogen assimilation, developmental activators (BrlA, AbaA, and WetA) and upstream transcription factor (FluG) required for conidiation, P-type N+/K+ ATPases (Ena1-5) required for intracellular N+/K+ homeostasis, and antioxidant enzymes involved in multiple stress responses. Our study unveils that the loop and four C-terminal Ser/Thr residues are all vital for the regulatory role of Ste7 in the growth, conidiation, virulence, and/or stress tolerance of B. bassiana and perhaps other filamentous fungi.