Surface wettability plays a critical role in a variety of key areas including orthopaedic implants and chemical engineering. Anisotropy in wettability can arise from surface grooves, which are of particular relevance to orthopaedic implants because they can mimic collagen fibrils that are the basic components of the extracellular matrix. Titanium (Ti) and its alloys have been widely used for orthopaedic and dental implant applications. This study is concerned with the fabrication of Ti-coated microgrooves with different groove widths and the characterisation of the anisotropy in wettability through measuring water contact angles, compared with both the Wenzel and Cassie models. Experimental results revealed that there existed significant anisotropy in the wettability of Ti-coated microgrooves, and the degree of anisotropy (Δθ) increased with an increasing groove width from 5 μm to 20 μm. On average, the contact angle measured parallel to the groove direction (θ//) was about 50°-60° smaller than that measured perpendicular to the groove direction (θa). In general, the Wenzel model predicted the contact angles along the surface groove direction reasonably, and so did the Cassie model for the contact angles perpendicular to the groove direction. Osteoblast spreading was affected by the anisotropy in wettability, which occurred preferably along, rather than perpendicular to, the groove direction. These findings are informative for the design of Ti implant surfaces when anisotropy in wettability matters.