Mechanical stress is an important regulatory factor in bone homeostasis. Mechanical stimulation of osteoblasts has been shown to elicit an increase in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i). The pattern of functional expression of mechanosensitive ion channels remains unclear, however. Therefore, the purpose of this study was to investigate the pharmacological characteristics of [Ca²⁺]_i in response to direct mechanical stimulation in osteoblasts. The morphological expression of mechanosensitive ion channels was also examined. Mouse osteoblast-like cells (MC3T3-E1 cells) were loaded with fura-2-acetoxymethyl ester, after which [Ca²⁺]_i was measured. Increased levels of [Ca²⁺]_i were observed in MC3T3-E1 cells in response to direct mechanical stimulation by means of a glass micropipette, but no desensitization. Application of a hypotonic solution also induced an increase in [Ca²⁺]_i but was accompanied by a desensitizing effect. Extracellular Gd³⁺, GsMTx4, or RN-1734 reversibly inhibited this mechanical stimulation-induced increase in [Ca²⁺]_i, whereas no inhibitory effect was observed with HC030031 or clemizole. When osteoblasts were stimulated with Yoda1, an increase was observed in [Ca²⁺]_i together with a significant desensitizing effect. Immunoreactivity against Piezo1 and TRPV4 channel antibodies was detected in MC3T3-E1 cells. These results suggest that osteoblasts express Piezo1 and TRPV4 channels, which are involved in mechanosensitive processes during mechanical stress.