We investigated the effects of alkaline pH on developing osteoblasts. Cells of the osteoblast-like cell line MC3T3-E1 were initially cultured for six days in HEPES-buffered media with pH ranging from 7.2 to 9.0. Cell count, cellular WST-1 metabolism, and ATP content were analyzed. The three parameters showed a pH optimum around pH 8.4, exceeding the recommended buffer range of HEPES at the alkaline flank. Therefore, only pH 7.2, 7.4, 7.8, and 8.4 media were used in more elaborate, daily investigations to reduce the effects of pH change within the pH control intervals of 24 h. All parameters exhibited similar pH behaviors, roughly showing increases to 130% and 230% at pH 7.8 and 8.4, as well as decreases to 70% at pH 7.2 when using the pH 7.4 data for reference. To characterize cell differentiation and osteoblastic cell function, cells were cultured at pH 7.4 and under alkaline conditions at pH 7.8 and 8.4 for 14 days. Gene expression and mineralization were evaluated using microarray technology and Alizarin staining. Under alkaline conditions, ATF4, a regulator for terminal differentiation and function as well as DMP1, a potential marker for the transition of osteoblasts into osteocytes, were significantly upregulated, hinting at an accelerated differentiation process. After 21 days, significant mineralization was only detected at alkaline pH. We conclude that elevated pH is beneficial for the cultivation of bone cells and may also provide therapeutic value in bone regeneration therapies.