This study assessed knowledge, attitude and practice (KAP) related to vitamin D and its relationship with vitamin D status among Malay female office workers. A total of 147 women aged between 20 and 55 years were recruited from a university in Kuala Lumpur. They answered questionnaires related to KAP on vitamin D, sun exposure, dietary vitamin D intake and physical activity. Serum 25-hydroxyvitamin D (25OHD) was analysed using an enzyme-linked immunoassay. Nearly half (45%) of the subjects had good knowledge but moderate attitude (76%) and practice (84%) towards sunlight exposure and dietary vitamin D intake. Median serum 25OHD was 34.1 nmol/L with the majority (91%) had vitamin D insufficiency (25OHD < 50 nmol/L). Knowledge was weakly associated with attitude (r = 0.29, p < 0.001) but no association was found between knowledge and practice (r = 0.08, p = 0.355) nor attitude and practice (r = -0.001, p = 0.994). Serum 25OHD was positively associated with sunlight exposure (r = 0.22, p = 0.008) and dietary vitamin D intake (r = 0.37, p < 0.001). It can be implied that this group is at increased risk of low bone health status, which highlights the needs of public health campaigns to improve their vitamin D status.